Personalized Mapping of Drug Metabolism by the Human Gut Microbiome.

The human gut microbiome harbors hundreds of bacterial species with diverse biochemical capabilities. Dozens of drugs have been shown to be metabolized by single isolates from the gut microbiome, but the extent of this phenomenon is rarely explored in the context of microbial communities. Here, we develop a quantitative experimental framework for mapping the ability of the human gut microbiome to metabolize small molecule drugs: Microbiome-Derived Metabolism (MDM)-Screen. Included are a batch culturing system for sustained growth of subject-specific gut microbial communities, an ex vivo drug metabolism screen, and targeted and untargeted functional metagenomic screens to identify microbiome-encoded genes responsible for specific metabolic events. Our framework identifies novel drug-microbiome interactions that vary between individuals and demonstrates how the gut microbiome might be used in drug development and personalized medicine.

A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of GlcNAc.

The zwitterions phosphorylcholine (PC) and phosphoethanolamine (PE) are often found esterified to certain sugars in polysaccharides and glycoconjugates in a wide range of biological species. One such modification involves PC attachment to the 6-carbon of N-acetylglucosamine (GlcNAc-6-PC) in N-glycans and glycosphingolipids (GSLs) of parasitic nematodes, a modification that helps the parasite evade host immunity. Knowledge of enzymes involved in the synthesis and degradation of PC and PE modifications is limited. More detailed studies on such enzymes would contribute to a better understanding of the function of PC modifications and have potential application in the structural analysis of zwitterion-modified glycans. In this study, we used functional metagenomic screening to identify phosphodiesterases encoded in a human fecal DNA fosmid library that remove PC from GlcNAc-6-PC. A novel bacterial phosphodiesterase was identified and biochemically characterized. This enzyme (termed GlcNAc-PDase) shows remarkable substrate preference for GlcNAc-6-PC and GlcNAc-6-PE, with little or no activity on other zwitterion-modified hexoses. The identified GlcNAc-PDase protein sequence is a member of the large endonuclease/exonuclease/phosphatase superfamily where it defines a distinct subfamily of related sequences of previously unknown function, mostly from Clostridium bacteria species. Finally, we demonstrate use of GlcNAc-PDase to confirm the presence of GlcNAc-6-PC in N-glycans and GSLs of the parasitic nematode Brugia malayi in a glycoanalytical workflow.

Integrating functional metagenomics to decipher microbiome-immune interactions.

Microbial metabolites can be viewed as the cytokines of the microbiome, transmitting information about the microbial and metabolic environment of the gut to orchestrate and modulate local and systemic immune responses. Still, many immunology studies focus solely on the taxonomy and community structure of the gut microbiota rather than its functions. Early sequencing-based microbiota profiling approaches relied on PCR amplification of small regions of bacterial and fungal genomes to facilitate identification of the microbes present. However, recent microbiome analysis methods, particularly shotgun metagenomic sequencing, now enable culture-independent profiling of microbiome functions and metabolites in addition to taxonomic characterization. In this review, we showcase recent advances in functional metagenomics methods and applications and discuss the current limitations and potential avenues for future development. Importantly, we highlight a few examples of key areas of opportunity in immunology research where integrating functional metagenomic analyses of the microbiome can substantially enhance a mechanistic understanding of microbiome-immune interactions and their contributions to health and disease states.

Cat and dog feces as reservoirs of diverse novel antibiotic resistance genes.

Companion animals have the potential to greatly enhance the physical and mental health of humans, thus leading to an increased focus on the interactions between humans and pets. Currently, the inappropriate and excessive utilization of antimicrobial agents has become prevalent in veterinary clinical practice for pets. This antibiotic contamination phenomenon has a profound impact on the enrichment of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in pets. However, the pet-associated resistome, especially the novel ARGs in pets, represents a relatively neglected area. In this study, we successfully constructed a total of 12 libraries using the functional metagenomics approach to assess the diversity of ARGs in pet cats and dogs from four pet hospitals. Through the integration of functional screening and high-throughput sequencing, a total of 122 antibiotic resistance determinants were identified, of which 15 were classified as putative novel ARGs originating from five classes. Functional assessment demonstrated that 6 novel ARGs including one ß-lactam, two macrolides, two aminoglycosides, and one rifamycin (RIF), namely blaPF, ermPF, msrPF, aac(6')PF, aph(3')PF, and arrPF, exhibited functionally activity in conferring bacterial phenotypic resistance by increasing the minimum inhibitory concentrations (MICs) with a 4- to 128-fold. Genetic context analysis demonstrated that, with the exception of aac(6')PF and arrPF, the remaining four novel ARGs were found adjacent to mobile genetic elements (MGEs) including IS elements or transposases, which provided a prerequisite for horizontal transfer of these novel ARGs, thereby offering an explanation for their detection in diverse samples collected from various sampling sites. The current study has unveiled the significant role of cat and dog feces as one source of reservoirs of diverse novel ARGs, while also highlighting the potential adverse consequences of their further spread to medically significant pathogens and human commensal organisms.

Characterization and predictive functional profiles on metagenomic 16S rRNA data of liver transplant recipients: A longitudinal study.

Long-term survival after Liver Transplantation (LT) is often compromised by infectious and metabolic complications. We aimed to delineate alterations in intestinal microbiome (IM) over time that could contribute to medical complications compromising long-term survival following LT. Fecal samples from LT recipients were collected at 3 months (3 M) and 6 months (6 M) post-LT. The bacterial DNA was extracted using E.Z.N.A. Stool DNA Kit and 16S rRNA gene sequencing at V4 hypervariable region was performed. DADA2 and Phyloseq was implemented to analyze the taxonomic composition. Differentially abundant taxa were identified by metagenomeSeq and LEfSe. Piphillin, an Inferred functional metagenomic analysis tool was used to study the bacterial functional content. For comparison, healthy samples were extracted from NCBI and analyzed similarly. The taxonomic & functional profiles of LT recipients were validated with metagenomic sequencing data from animals exposed to immunosuppressants using Venny. Our findings provide a new perspective on longitudinal increase in specific IM communities post-LT along with an increase in bacterial genes associated with metabolic and infectious disease.

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases.

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

Ultrahigh-Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay.

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis.

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans.

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.

Two-step functional screen on multiple proteinaceous substrates reveals temperature-robust proteases with a broad-substrate range.

To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9-11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. KEY POINTS: • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.

An uncharacterized protein from the metagenome with no obvious homology to known lipases shows excellent alkaline lipase properties and potential applications in the detergent industry.

A novel lipase, Lip486, which has no obvious homology with known lipases, was discovered using functional metagenomics technology. Phylogenetic tree analysis suggested that the enzyme belongs to a new subfamily called lipolytic enzyme family II. To explore the enzymatic properties, lip486 was expressed heterologously and efficiently in Escherichia coli. The recombinant enzyme displayed the highest activity on the substrate p-nitrophenyl caprate with a carbon chain length of 10, and its optimum temperature and pH were 53 °C and 8.0, respectively. The recombinant Lip486 showed good activity and stability in strong alkaline and medium-low-temperature environments. The results of compatibility and soaking tests showed that the enzyme had good compatibility with 4 kinds of commercial detergents, and an appropriate soaking time could further improve the enzyme activity. Oil stain removal test results for a cotton cloth indicated that the washing performance of commercial laundry detergent supplemented with Lip486 was further improved. In addition, as one of the smallest lipases found to date, Lip486 also has the advantages of high yield, good stability and easy molecular modification. These characteristics reflect the good application prospects for Lip486 in the detergent and other industries in the future.

Bacterial communities and functional genes stimulated during phenanthrene degradation in soil by bio-microcapsules.

In this study, a taxonomic and functional metagenomic method was used to investigate the difference produced between degrading bacteria immobilized in layer-by-layer assembly (LBL) microcapsules or not during the bioremediation of a soil polluted with phenanthrene (PHE). Bioaugmentation with LBL microcapsule immobilized degrading bacteria could result in different changes of native microbial communities, shifting the functional gene constructions of polluted soils. The LBL treatment enhanced PHE degradation (initial concentration of 100 mg kg-1 dry soil) by 60% after 25 d compared to the free bacteria (FB). The enhancing effect of PHE degradation produced by the LBL treatment was found to be significantly associated with some crucial phyla (e.g., Bacteroides, Gemmatimonadetes and Acidobacteria) and genera including Streptomyces, Ramlibacter, Mycobacterium, Phycicoccus, Gemmatirosa, Flavisolibacter, Micromonospora, Acid_Candidatus_Koribacter and Gemmatimonas. The main differences of functional metagenomics between LBL and FB treatments were observed in higher levels in metabolism of aromatic hydrocarbons and its related functions or enzymes in the former, e.g., membrane transport systems, binding, substrate transporter, cleavage enzymes, dehydrogenation, oxidase, esterase and glycosidase, greatly favoring PHE mineralization. Therefore, our results provide useful findings on understanding of how immobilization strategies can influence the taxonomic and functional gene composition in soils, as well as polycyclic aromatic hydrocarbons (PAH) degradation.

Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine.

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.

Bacterial diversity and functional metagenomics expounding the diversity of xenobiotics, stress, defense and CRISPR gene ontology providing eco-efficiency to Himalayan Hot Springs.

Sikkim is one of the bio-diverse states of India, which harbors diverse alkaline and sulfur rich hot springs in its vicinity. However, there is a dearth of data present in terms of microbial and its functional diversity as only a few hot springs have been studied in this area. Thus, in this regard, microbial and functional diversity of two hot springs by NGS, PLFA, and culture-independent approaches were carried out. PLFA and culture-dependent analysis was complementary as the Gram-positive bacteria were abundant in both the hot springs with the dominance of phylum Firmicutes with Geobacillus. Metagenomic analysis revealed the abundance of Proteobacteria, Actinobacteria, and Firmicutes in both hot springs. Functional metagenomics suggested that both Yumthang and Reshi hot spring possess a diverse set of genes analogous to stress such as genes allied to osmotic, heat shock, and acid stresses; defense analogies such as multidrug resistance efflux pump, multidrug transport system, and ß-lactamase; and CRISPR analogues such as related to Cas1, Cas2, Cas3, cmr1-5 proteins, CT1972, and CT1133 gene families. The xenobiotic analogues were found against benzoate, nitrotolune, xylene, DDT, and chlorocyclohexane/chlorobenzene degradation. Thus, these defensive mechanisms against environmental and anthropogenic hiccups and hindrances provide the eco-efficiency to such thermal habitats. The higher enzymatic, degradation, defense, stress potential and the lower percentage identity (< 95%) of isolates encourage the further exploration and exploitation of these habitats for industrial and biotechnological purposes.

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156).

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.

Characterization of the circulating microbiome in acute-on-chronic liver failure associated with hepatitis B.

BACKGROUND: Patients with hepatitis B-related acute-on-chronic liver failure (HB-ACLF) may have an increased circulating microbial burden. This study aimed to assess circulating microbial load and composition and to explore the association between the circulating microbiome and both systemic inflammation (SI) and clinical outcome in HB-ACLF. METHODS: Plasma from 50 HB-ACLF patients, 23 healthy controls and 25 patients with compensated liver cirrhosis (C-LC) was analysed for chemokines/cytokines and bacterial DNA and further analysed by 16S rDNApyrosequencing. Linear discriminant analysis effect size (LEfSe) and inferred metagenomics analyses were performed. RESULTS: The circulating bacterial DNA was significantly increased in HB-ACLF patients compared to that in the control groups. The overall microbial diversity was significantly decreased in HB-ACLF patients. HB-ACLF patients were enriched with Moraxellaceae, Sulfurovum, Comamonas and Burkholderiaceae but were depleted in Actinobacteria, Deinococcus-Thermus, Alphaproteobacteria, Xanthomonadaceae and Enterobacteriaceae compared to controls. Network analysis revealed a direct positive correlation between Burkholderiaceae and chemokine IP-10 in HB-ACLF patients. The relative abundance of Prevotellaceae independently predicted 28-day mortality. Inferred functional metagenomics predicted an enrichment of bacteria with genes related to methane, alanine, aspartate, glutamate, pyrimidine, purine and energy metabolism. CONCLUSIONS: HB-ACLF patients display increased circulating microbial burden, altered microbiome composition and a shift in microbiome functionality. The alteration in circulating microbiota is associated with SI and clinical outcome in HB-ACLF.

A-GAME: improving the assembly of pooled functional metagenomics sequence data.

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. A-GAME is publicly available at http://beaconlab.it/agame.

Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning.

Activity-based screening of metagenomic DNA libraries is a promising approach to fish out genes encoding novel bioactive compounds/enzymes of industrial importance. The starting point of such functional screening in fosmid vectors is isolation of high molecular weight (HMW) DNA of sufficient purity from diverse environments. Metagenomic DNA isolation protocols mostly employ mechanical cell lysis that yields fragmented DNA. Those established for HMW DNA using enzymatic lysis have not considered samples with high lignocellulose or humic acid content. Enzymes from such environments are in great demand for bioenergy, paper, and related industries. Thus, an improved method was standardized that has three key features, i.e., use of harvested microbial biomass instead of raw samples, removal of humic substances prior to cell lysis by aluminum sulfate flocculation, and enzymatic/chemical lysis of cells with a lysozyme, mutanolysin, proteinase K, and SDS cocktail followed by phenol-chloroform extraction and precipitation of DNA by polyethylene glycol and NaCl. HMW DNA (~ 40 kb) was efficiently isolated from garden and forest soils, rice straw compost, and degrading wood from a hypersaline lake. The humic acid removal efficiency across samples was 96-98%. The isolated DNA was of high quality/purity and could be successfully used in downstream applications like PCR, ligation, and fosmid cloning. In fact, the DNA was directly used without any size selection, for fosmid library preparation with 70-90% efficiency as compared to the control insert. Thus, the method could suitably be used for HMW DNA isolation for the functional screening of enzymes from diverse humic acid-/lignocellulose-rich environments.

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist.

The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. Functional metagenomics provides a systematic means of surveying commensal DNA for genes that encode effector functions. Here, we examine 3,000 Mb of metagenomic DNA cloned from three phenotypically distinct patients for effectors that activate NF-κB, a transcription factor known to play a central role in mediating responses to environmental stimuli. This screen led to the identification of 26 unique commensal bacteria effector genes (Cbegs) that are predicted to encode proteins with diverse catabolic, anabolic, and ligand-binding functions and most frequently interact with either glycans or lipids. Detailed analysis of one effector gene family (Cbeg12) recovered from all three patient libraries found that it encodes for the production of N-acyl-3-hydroxypalmitoyl-glycine (commendamide). This metabolite was also found in culture broth from the commensal bacterium Bacteroides vulgatus, which harbors a gene highly similar to Cbeg12. Commendamide resembles long-chain N-acyl-amides that function as mammalian signaling molecules through activation of G-protein-coupled receptors (GPCRs), which led us to the observation that commendamide activates the GPCR G2A/GPR132. G2A has been implicated in disease models of autoimmunity and atherosclerosis. This study shows the utility of functional metagenomics for identifying potential mechanisms used by commensal bacteria for host interactions and outlines a functional metagenomics-based pipeline for the systematic identification of diverse commensal bacteria effectors that impact host cellular functions.

New Bacterial Phytase through Metagenomic Prospection.

Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. ß-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for ß-propeller phytase functions could be of great benefit to biotechnology.