Sequence-based Metagenomics Research Articles

Recent Advances in Metagenomic Approaches, Applications, and Challenge.

Advances in metagenomics analysis with the advent of next-generation sequencing have extended our knowledge of microbial communities as compared to conventional techniques providing advanced approach to identify novel and uncultivable microorganisms based on their genetic information derived from a particular environment. Shotgun metagenomics involves investigating the DNA of the entire community without the requirement of PCR amplification. It provides access to study all genes present in the sample. On the other hand, amplicon sequencing targets taxonomically important marker genes, the analysis of which is restricted to previously known DNA sequences. While sequence-based metagenomics is used to analyze DNA sequences directly from the environment without the requirement of library construction and with limited identification of novel genes and products that can be complemented by functional genomics, function-based metagenomics requires fragmentation and cloning of extracted metagenome DNA in a suitable host with subsequent functional screening and sequencing clone for detection of a novel gene. Although advances were made in metagenomics, different challenges arise. This review provides insight into advances in the metagenomic approaches combined with next-generation sequencing, their recent applications highlighting the emerging ones, such as in astrobiology, forensic sciences, and SARS-CoV-2 infection diagnosis, and the challenges associated. This review further discusses the different types of metagenomics and outlines advancements in bioinformatics tools and their significance in the analysis of metagenomic datasets.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, β-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

The renewable, abundant, and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples’ microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited β-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Key points• Sequence- and function-based metagenomics were used to find CAZymes in compost samples.• Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes.• Compost-derived fosmid libraries are enriched in clones with β-glucosidase activity.

Novel bacteriophage-mediated β-lactamase-encoding genes and their risk assessment in environmental communities

Bacteria can obtain antibiotic resistance genes (ARGs) through multiple transmission routes, including horizontal gene transfer mediated by phages. Sequence-based metagenomics can detect novel phage-derived ARGs and avoid spending a great deal of time on phage isolation. Here, a metagenomic method was applied to explore the presence of ARGs in phages in freshwater samples. Two novel β-lactamase-encoding genes, blaORF88 and blaORF47, were identified and characterized in phage genomes for the first time in this study. Phylogenetic analyses indicated that blaORF88 is a member of the class C β-lactamase family, while blaORF47 formed an independent branch away from the class C β-lactamases. The average absolute abundance of blaORF88 in phage DNA and bacterial DNA was as high as 5.14 log GC/100 ml and 5.73 log GC/ml, respectively. The blaORF47 gene detected in bacterial DNA was found at the highest concentration, particularly in urban sewage samples, with 5.32 log GC/ml. Our findings raise concerns that phages might carry unknown or novel ARGs, which may spread into bacteria, leading to the emergence of antibiotic-resistant bacteria in the environment, and we urgently need effective strategies to avoid the health risks caused by ARGs.

Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functional CopA in Environmental Microbiomes

Exploring the natural diversity of functional genes/proteins from environmental DNA in high throughput remains challenging. In this study, we developed a sequence-based functional metagenomics procedure for mining the diversity of copper (Cu) resistance gene copA in global microbiomes, by combining the metagenomic assembly technology, local BLAST, evolutionary trace analysis (ETA), chemical synthesis, and conventional functional genomics. In total, 87 metagenomes were collected from a public database and subjected to copA detection, resulting in 93,899 hits. Manual curation of 1214 hits of high confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually, 175 novel copA sequences of high quality were discovered. Phylogenetic analysis showed that almost all these putative CopA proteins were distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known copA genes were chemically synthesized for further functional genomic tests using the Cu-sensitive Escherichia coli (ΔcopA). The growth test and Cu uptake determination showed that five novel clones had positive effects on host Cu resistance and uptake. One recombinant harboring copA-like 15 (copAL15) successfully restored Cu resistance of the host with a substantially enhanced Cu uptake. Two novel copA genes were fused with the gfp gene and expressed in E. coli for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localized to the membrane. The results here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.

Dysbiosis of Gut Microbiota in Patients Undergoing Cardiac Surgery

The diversity of bacteria, viruses, eukaryotic organisms, and archaea that live in the gastrointestinal tract and have coevolved with the host over thousands of years to establish a complex and advantageous relationship is referred to as the “gut microbiota” or “gut microbiome.” The numerous and diverse gut microbiota play an important role mostly in the betterment of human health by facilitating the breakdown of food to release nutrients that would otherwise be inaccessible to the host, encouraging host cells proliferation and differentiation, defending the host against pathogen colonization, and activating or modulating the immune system. The gut microbial community plays an important role in protecting the host against pathogenic microbes, modulating immunity, and regulating metabolic processes and is even regarded as an endocrine organ. The term “dysbiosis” refers to the imbalance of the gut bacterial microbiota, which has been linked to several disorders such as cardiovascular diseases, obesity, irritable bowel syndrome, colorectal cancer, and sepsis. During cardiac surgery, intestinal ischemia-reperfusion causes an inflammatory response throughout the body. Since the intestines are an organ that is vulnerable to ischemia, germs and endotoxins can move between organs. As a consequence, the gut leads to sepsis after surgery. Next-generation sequencing technologies have enabled analysis of a large number of microorganisms. Metagenomics can be used to study intestinal microbiome diversity and dysbiosis, as well as its relationship to health and disease. Sequence-based and multifunctional metagenomics will also provide insights that will lead to greater knowledge than it is ever been of the structural and functional microbiomes.

Discovery of novel enzymes from extreme environments through metagenomic approaches: A mini-review

Extremophilic microorganisms are able to survive under extreme conditions of temperature, pH, salinity and pressure, such as hypersaline soils, hydrothermal vents, acidic coal mines and soda lakes. These microorganisms have special genetic and physiological modifications to survive under abiotic and biotic stresses. Extremozymes are considered as an important source for different industrial and biotechnological applications. With the advent of meta-omics approaches, the discovery of novel enzymes has accelerated. Using function- and sequence-based metagenomics, a large number of enzymes have been identified and characterized directly from extreme environments. Many extremozymes such as proteases, amylases, esterases, cellulases, chitinases, oxidoreductases and hydrolases have special properties to work at a wide range of salt, pH and temperature. In this review, the discovery and industrial applications of novel enzymes from extreme environments through metagenomic approaches have been discussed. We have also explained the screening methods of novel extremozymes through function- and sequence-based metagenomics.

Fermentation microbiome and metabolic profiles of Indian palm wine

Fermented food and beverages provide huge benefits to humans. The nutritionally rich palm wine (toddy) is a popular fermented beverage in India. The present study reveals the bacterial diversity and metabolic profile of Indian palm wine using next-generation sequencing of the V3-V4 regions of the 16S rRNA gene. The metagenomic sequencing reveals the dominance of the phyla Proteobacteria, Firmicutes, and Tenericutes in the Indian palm wine. Bacteria such as Acetobacter, Lactobacillus, Candidatus phytoplasma, and Gluconobacter were abundant in palm wine. The sequence-based metagenomics approach revealed that Acetobacter sp. and Lactobacilli sp., which are beneficial bacteria, were the most predominant microbial populations in the Indian palm wine. The metabolic functional profile of the palm wine microbiome was studied based on gene ontology terms to identify industrial by-products and bio-prospecting genes. Metabolic profiling revealed that a major microbial population was involved in ammonia oxidation, sulfate reduction, nitrite reduction, atrazine metabolization, and acid production. The results can aid in developing probiotics from palm wine for human consumption with a multitude of health benefits.

Metagenomics: A Novel Tool for Livestock and Poultry Improvement: A Review

A distinct mechanism of generating metagenome is metagenomics which includes sequencing the whole DNA extracted from an environmental sample, mapping it to a reference database followed by gene annotation. Advancement in sequence-based metagenomics has significantly reduced processing costs and has acquired a rapid pace. Metagenomics has been crucial in investigating “hidden” genetic characteristics and construction biotechnological processes through the discovery of novel genes, enzymes, pathways and bioactive molecules with entirely different or improved biochemical functions. Metagenomics is a fairly recent addition to the molecular toolbox and is the simplest, impartial way of challenging the adaptive ability of microbial populations. Metagenomics is beneficial in recognising the complex consortium of bacteria, protozoa, archaea, fungi, etc. and association amongst them resulting in higher feed utilization and productivity of animals. It allows formulating probiotics feed materials, as well as in immunomodulation in both livestock and poultry. This review emphasizes significant recent achievements in metagenomics, offers insights into the possibilities, modern-day challenges and its utility in livestock and poultry.

Bioprospecting of Novel Extremozymes From Prokaryotes-The Advent of Culture-Independent Methods.

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance. Genetic and structural information derived from extremophiles and extremozymes can be used for bioengineering other nontolerant enzymes. Furthermore, extremophiles can be a valuable resource for novel biotechnological and biomedical products due to their biosynthetic properties. However, understanding life under extreme conditions is challenging due to the difficulties of in vitro cultivation and observation since > 99% of organisms cannot be cultivated. Consequently, only a minor percentage of the potential extremophiles on Earth have been discovered and characterized. Herein, we present a review of culture-independent methods, sequence-based metagenomics (SBM), and single amplified genomes (SAGs) for studying enzymes from extremophiles, with a focus on prokaryotic (archaea and bacteria) microorganisms. Additionally, we provide a comprehensive list of extremozymes discovered via metagenomics and SAGs.

Functional Metagenomics from the Rumen Environment—A Review

The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunity. A better understanding of rumen microbial processes and identification of which populations are responsible for specific functions within the rumen microbiome will lead to better management and sustainable utilization of the available feed base while maintaining a low environmental impact. Recent advance in the culture independent method of microbiology such as metagenomics, unravels potentially the rumen microbial process. There are two basic types of metagenomics studies: Sequence-based and function-based metagenomics. Sequence-based metagenomics involves sequencing and analysis of DNA from environmental samples. Its purpose is to assemble genomes, identify genes, find complete metabolic pathways, and compare organisms of different communities. Whereas functional metagenomics is the study of the collective genome of a microbial community by expressing it in a foreign host usually Escherichia coli (E. coli). It is a promising approach unearthing novel enzymes even from yet to culture rumen microbiota. Further advances in the screening techniques promise vast opportunities to rumen microbiologists, and animal nutritionist. The identification of novel enzyme through functional metagenomics consists of three parts: rumen sample collection; DNA library construction and screening of individual clone. Functional metagenomics was successfully applied to identify different antibiotics, hydrolytic enzymes, antibiotic resistance genes, and many other functions; moreover, it allowed characterization of genes encoding enzymes with a particular activity, which represents completely novel sequence. There are a number of outputs from functionally screened rumen product such as carbohydrate active enzymes (CAZymes) that can break down plant cell walls. Company involved commercialization of metagenomics research such as Syngenta, Genencor International, BRAIN etc., has produced many biological molecules of commercial interest. The aim of this paper is to elucidate functional metagenomics, from rumen environment and its potential for commercial purpose.

β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. β-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding β-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with β-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-β-galactoside and lactose. The activity levels of one of these novel β-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic β-galactosidases from diary stabilization ponds’ metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries’ by-products.

A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

Assessment of Gram- and Viability-Staining Methods for Quantifying Bacterial Community Dynamics Using Flow Cytometry.

Over the past years, gut microbiota became a major field of interest with increasing reports suggesting its association with a large number of human diseases. In this context, there is a major interest to develop analysis tools allowing simple and cost-effective population pattern analysis of these complex ecosystems to follow changes over time. Whereas sequence-based metagenomics profiling is widely used for microbial ecosystems characterization, it still requires time and specific expertise for analysis. Flow cytometry overcomes these disadvantages, providing key information on communities within hours. In addition, it can potentially be used to select, isolate and cultivate specific bacteria of interest. In this study, we evaluated the culturability of strictly anaerobic bacteria that were stained with a classical Live/Dead staining, and then sorted using flow cytometry under anaerobic conditions. This sorting of “viable” fraction demonstrated that 10–80% of identified “viable” cells of pure cultures of strictly anaerobic bacteria were culturable. In addition, we tested the use of a combination of labeled vancomycin and Wheat Germ Agglutinin (WGA) lectin to discriminate Gram-positive from Gram-negative bacteria in complex ecosystems. After validation on both aerobic/anaerobic facultative and strictly anaerobic bacteria, the staining methods were applied on complex ecosystems, revealing differences between culture conditions and demonstrating that minor pH variations have strong impacts on microbial community structure, which was confirmed by 16S rRNA gene sequencing. This combination of staining methods makes it possible to follow-up evolutions of complex microbial communities, supporting its future use as a rapid analysis tool in various applications. The flow cytometry staining method that was developed has the potential to facilitate the analysis of complex ecosystems by highlighting changes in bacterial communities’ dynamics. It is assumed to be applicable as an efficient and fast approach to improve the control of processes linked to a wide range of ecosystems or known communities of bacterial species in both research and industrial contexts.

Recombinant production and characterization of a novel esterase from a hypersaline lake, Acıgöl, by metagenomic approach.

The aim of this study was to isolate a novel esterase from a hypersaline lake by sequence-based metagenomics. The metagenomic DNA was isolated from the enriched hypersaline lake sediment. Degenerate primers targeting the conserved regions of lipolytic enzymes of halophilic microorganisms were used for polymerase chain reaction (PCR) and a whole gene was identified by genome walking. The gene was composed of 783bp, which corresponds to 260 amino acids with a molecular weight of 28.2kDa. The deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. Recombinantly expressed enzyme exhibited maximum activity towards pNP-hexanoate with a kcat value of 12.30s-1. The optimum pH and temperature of the enzyme were found as 9 and 30°C, respectively. The effects of NaCl, solvents, metal ions, detergents and enzyme inhibitors were also studied. In conclusion, a novel enzyme, named as hypersaline lake "Acıgöl" esterase (hAGEst), was identified by sequence-based metagenomics. The high expression level, the ability to maintain activity at cold temperatures and tolerance to DMSO and metal ions are the most outstanding properties of the hAGEst.

Metagenomic analysis of isolation methods of a targeted microbe, Campylobacter jejuni, from chicken feces with high microbial contamination

BackgroundOriginating from poultry, particularly chickens, Campylobacter jejuni is the leading foodborne pathogen worldwide and a major cause of campylobacteriosis. Isolating C. jejuni is difficult due to its specific growth requirements, the presence of viable but non-culturable bacteria, and because it is often masked by competing flora. Currently, there is no optimized method for isolating C. jejuni from chicken feces. Here, we evaluated the method for isolating C. jejuni from chicken feces using culture-independent sequence-based metagenomics and culture-dependent tools. Further, we assessed changes in microbial communities during microbe isolation to determine how the process can be improved.ResultsFourteen different variations of C. jejuni isolation procedures were applied to all 35 chicken fecal samples. These variations included using different enrichment broths (without enrichment or enrichment in Bolton or Preston broth), different ratios of sample-to-enrichment broth (1:101, 1:102, and 1:103), and different selective agars (modified charcoal-cefoperazone-deoxycholate agar (mCCDA) or Preston agar). Enrichment during isolation of C. jejuni was evaluated on the basis of microbial diversity and taxonomic composition using metagenomics tools. The effect of selective media was evaluated using a combination of metagenomics and culture-dependent tools. Microbial diversity significantly decreased during the enrichment process, regardless of the type of enrichment broth, with the most significant decrease observed at a feces-to-broth ratio of 1:103. Particularly, in 103-Preston broth, the relative abundance of Campylobacter increased, while extended-spectrum beta-lactamase-producing Escherichia coli, which interfere with Campylobacter isolation, decreased. Metagenomics results were validated by quantitative PCR and culture-dependent analysis. Additionally, selective media affected the isolation results, although microbes with high relative abundance during enrichment were also frequently isolated using culture-dependent methods. Significantly more C. jejuni was isolated from mCCDA than from Preston agar enriched in 103 Preston broth.ConclusionsEnrichment in Preston broth at a ratio of 1:103 followed by spreading onto mCCDA was the most effective method for isolating C. jejuni. This is the first study to apply metagenomics to evaluate a method for isolating a targeted microbe, C. jejuni, from chicken feces, a source with high microbial contamination. Thus, metagenomics can be applied to improve methods for isolating bacteria that are difficult to separate.

Screening novel β-galactosidases from a sequence-based metagenome and characterization of an alkaline β-galactosidase for the enzymatic synthesis of galactooligosaccharides

βgalactosidases have wide industrial applications in lactose hydrolysis and transglycosylation reactions. Therefore, there is a need to mine novel and high-quality β-galactosidases with good tolerance and novel features from harsh environments and genomic databases. In this study, an Escherichia coli β-galactosidase-deficient host, ΔlacZ(DE3)pRARE, was constructed by the CRISPR-Cas9 system for screening active β-galactosidases. Of thirty selected β-galactosidases, twelve novel enzymes showed β-galactosidase activity, four of which were purified for further study. BGal_375 exhibited maximal activity at pH 8 and 50 °C. The concentrations of two types of galactooligosaccharides, tri- and tetra-saccharides, produced by BGal_375, reached 64.53 g/l and 8.32 g/l, respectively. BGal_375 displayed a Km value of 1.65 mM and kcat value of 53 s−1 for p-nitrophenyl-β-d-galactopyranoside (pNPG). BGal_137, BGal_144-3, and BGal_145-2 showed promising hydrolytic activity for pNPG. BGal_137 is a homodimer while BGal_144-3, BGal_145-2, and BGal_375 were all monomeric. This study provided an efficient solution for the identification of new β-galactosidases from metagenomic data, and an alkaline β-galactosidase efficient for the synthesis of galactooligosaccharides was obtained, which is important for potential industrial applications.

Exploring the Impacts of Anthropogenic Disturbance on Seawater and Sediment Microbial Communities in Korean Coastal Waters Using Metagenomics Analysis

The coastal ecosystems are considered as one of the most dynamic and vulnerable environments under various anthropogenic developments and the effects of climate change. Variations in the composition and diversity of microbial communities may be a good indicator for determining whether the marine ecosystems are affected by complex forcing stressors. DNA sequence-based metagenomics has recently emerged as a promising tool for analyzing the structure and diversity of microbial communities based on environmental DNA (eDNA). However, few studies have so far been performed using this approach to assess the impacts of human activities on the microbial communities in marine systems. In this study, using metagenomic DNA sequencing (16S ribosomal RNA gene), we analyzed and compared seawater and sediment communities between sand mining and control (natural) sites in southern coastal waters of Korea to assess whether anthropogenic activities have significantly affected the microbial communities. The sand mining sites harbored considerably lower levels of microbial diversities in the surface seawater community during spring compared with control sites. Moreover, the sand mining areas had distinct microbial taxonomic group compositions, particularly during spring season. The microbial groups detected solely in the sediment load/dredging areas (e.g., Marinobacter, Alcanivorax, Novosphingobium) are known to be involved in degradation of toxic chemicals such as hydrocarbon, oil, and aromatic compounds, and they also contain potential pathogens. This study highlights the versatility of metagenomics in monitoring and diagnosing the impacts of human disturbance on the environmental health of marine ecosystems from eDNA.

Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool.

SummaryThe advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence‐based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3′‐aminoglycoside phosphotransferase [APH(3′)] and a class A beta‐lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3′) was active in vivo. Interestingly, APH(3′) proved to be thermostable (T m = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T m = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.