Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics.

One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and post-translational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for function-driven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

Metagenomics of Atacama Lithobiontic Extremophile Life Unveils Highlights on Fungal Communities, Biogeochemical Cycles and Carbohydrate-Active Enzymes.

Halites, which are typically found in various Atacama locations, are evaporitic rocks that are considered as micro-scaled salterns. Both structural and functional metagenomic analyses of halite nodules were performed. Structural analyses indicated that the halite microbiota is mainly composed of NaCl-adapted microorganisms. In addition, halites appear to harbor a limited diversity of fungal families together with a biodiverse collection of protozoa. Functional analysis indicated that the halite microbiome possesses the capacity to make an extensive contribution to carbon, nitrogen, and sulfur cycles, but possess a limited capacity to fix nitrogen. The halite metagenome also contains a vast repertory of carbohydrate active enzymes (CAZY) with glycosyl transferases being the most abundant class present, followed by glycosyl hydrolases (GH). Amylases were also present in high abundance, with GH also being identified. Thus, the halite microbiota is a potential useful source of novel enzymes that could have biotechnological applicability. This is the first metagenomic report of fungi and protozoa as endolithobionts of halite nodules, as well as the first attempt to describe the repertoire of CAZY in this community. In addition, we present a comprehensive functional metagenomic analysis of the metabolic capacities of the halite microbiota, providing evidence for the first time on the sulfur cycle in Atacama halites.

Sex-Specific Linkages Between Taxonomic and Functional Profiles of Tick Gut Microbiomes.

Ticks transmit the most diverse array of disease agents and harbor one of the most diverse microbial communities. Major progress has been made in the characterization of the taxonomic profiles of tick microbiota. However, the functional profiles of tick microbiome have been comparatively less studied. In this proof of concept we used state-of-the-art functional metagenomics analytical tools to explore previously reported datasets of bacteria found in male and female Ixodes ovatus, Ixodes persulcatus, and Amblyomma variegatum. Results showed that both taxonomic and functional profiles have differences between sexes of the same species. KEGG pathway analysis revealed that male and female of the same species had major differences in the abundance of genes involved in different metabolic pathways including vitamin B, amino acids, carbohydrates, nucleotides, and antibiotics among others. Partial reconstruction of metabolic pathways using KEGG enzymes suggests that tick microbiome form a complex metabolic network that may increase microbial community resilience and adaptability. Linkage analysis between taxonomic and functional profiles showed that among the KEGG enzymes with differential abundance in male and female ticks only 12% were present in single bacterial genera. The rest of these enzymes were found in more than two bacterial genera, and 27% of them were found in five up to ten bacterial genera. Comparison of bacterial genera contributing to the differences in the taxonomic and functional profiles of males and females revealed that while a small group of bacteria has a dual-role, most of the bacteria contribute only to functional or taxonomic differentiation between sexes. Results suggest that the different life styles of male and female ticks exert sex-specific evolutionary pressures that act independently on the phenomes (set of phenotypes) and genomes of bacteria in tick gut microbiota. We conclude that functional redundancy is a fundamental property of male and female tick microbiota and propose that functional metagenomics should be combined with taxonomic profiling of microbiota because both analyses are complementary.

Expanding the Toolbox of Broad Host-Range Transcriptional Terminators for Proteobacteria through Metagenomics.

As the field of synthetic biology moves toward the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacterium Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria, for instance Pseudomonas putida. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in P. putida. Further characterization allowed the identification of 4 unique sequences ranging from 58 to 181 bp long that efficiently terminate transcription in P. putida, E. coli, Burkholderia phymatum, and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated ß-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome.

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel ß-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel ß-glucosidase - named Lfa2 - displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing ß-glucose, ß-galactose, ß-xylose, ß-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, ß-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that ß-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

Distinctive Archaeal Composition of an Artisanal Crystallizer Pond and Functional Insights Into Salt-Saturated Hypersaline Environment Adaptation.

Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cáhuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cáhuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cáhuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them.

Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli.

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we experimentally mined novel constitutive promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Through the expression profiling of fluorescent clones from two independent soil sample libraries, we have analyzed the regulatory dynamics of 260 clones with candidate promoters as a set of active metagenomic promoters in the host Escherichia coli. Through an in-depth analysis of selected clones, we were able to further explore the architecture of metagenomic fragments and to report the presence of multiple promoters per fragment with a dominant promoter driving the expression profile. These approaches resulted in the identification of 33 novel active promoters from metagenomic DNA originated from very diverse phylogenetic groups. The in silico and in vivo analysis of these individual promoters allowed the generation of a constitutive promoter consensus for exogenous sequences recognizable by E. coli in metagenomic studies. The results presented here demonstrates the potential of functional metagenomics for exploring environmental bacterial communities as a source of novel regulatory genetic parts to expand the toolbox for microbial engineering.

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.

Early bacterial biofilm colonizers in the coastal waters of Mauritius

Background: The past years have witnessed a growing number of researches in biofilm forming communities due to their environmental and maritime industrial implications. To gain a better understanding of the early bacterial biofilm community, microfiber nets were used as artificial substrates and incubated for a period of 24 h in Mauritian coastal waters. Next-generation sequencing technologies were employed as a tool for identification of early bacterial communities. Different genes associated with quorum sensing and cell motility were further investigated. Results: Proteobacteria were identified as the predominant bacterial microorganisms in the biofilm within the 24 h incubation, of which members affiliated to Gammaproteobacteria, Alphaproteobacteria and Betaproteobacteria were among the most abundant classes. The biofilm community patterns were also driven by phyla such as Firmicutes, Bacteroidetes, Chloroflexi, Actinobacteria and Verrucomicrobia. The functional analysis based on KEGG classification indicated high activities in carbohydrate, lipid and amino acids metabolism. Different genes encoding for luxI, lasI, agrC, flhA, cheA and cheB showed the involvement of microbial members in quorum sensing and cell motility. Conclusion: This study provides both an insight on the early bacterial biofilm forming community and the genes involved in quorum sensing and bacterial cell motility.

Functional Metagenomics as a Tool for Identification of New Antibiotic Resistance Genes from Natural Environments.

Antibiotic resistance has become a major concern for human and animal health, as therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. The problem is compounded by low investment in antibiotic research and lack of new effective antimicrobial drugs on the market. Exploring environmental antibiotic resistance genes (ARGs) will help us to better understand bacterial resistance mechanisms, which may be the key to identifying new drug targets. Because most environment-associated microorganisms are not yet cultivable, culture-independent techniques are essential to determine which organisms are present in a given environmental sample and allow the assessment and utilization of the genetic wealth they represent. Metagenomics represents a powerful tool to achieve these goals using sequence-based and functional-based approaches. Functional metagenomic approaches are particularly well suited to the identification new ARGs from natural environments because, unlike sequence-based approaches, they do not require previous knowledge of these genes. This review discusses functional metagenomics-based ARG research and describes new possibilities for surveying the resistome in environmental samples.

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor.

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h(-1) OD(-1) L(-1).

A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues.

Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol).

Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures.