Metagenomic approach to identify genes encoding for glycoside hydrolases in composting samples

Metagenomics involves the study of the genomic DNA from a set of microorganisms present in a particular environmental sample. This approach has emerged as a promising culture-independent technique to explore the diversity and function of microbiomes, allowing the discovery of novel biochemical compounds, namely enzymes with high potential for industrial applications. Composting habitats are characterized by a high microbial diversity, and represent a suitable source of robust enzymes able to convert the recalcitrant structure of lignocellulose, such as cellulases, endohemicellulases, oligosaccharide-degrading enzymes, and debranching enzymes. In fact, several lignocellulose-degrading enzymes have been successfully identified in composting samples following metagenomic approaches. The efficient handling, processing, and analysis of the large metagenomic datasets generated by next-generation sequencing platforms can be achieved using advanced bioinformatics pipelines. In this work, composting samples were collected from three Portuguese composting units, which handle different types of wastes. The metagenomic DNA was extracted from the composting samples, the three composting metagenomes were analyzed by shotgun sequencing and a comparative analysis was performed between our samples and composting samples selected from the literature to evaluate the potential of these environments for lignocellulosic biomass conversion. The metagenomic sequencing data from all samples were processed using appropriate bioinformatics tools and the functional annotation of genes encoding glycoside hydrolases was carried out using the CAZy database. Our bioinformatics pipeline revealed that all samples were enriched in cellulases, endoglucanases, and -glucosidases, which confirms the richness of composting habitats, regardless of waste compositions, in lignocellulose-degrading enzymes. As these compost samples were collected in the thermophilic phase, the identified enzymes may harbor interesting features for industrial purposes, including catalytic activity under high temperatures.info:eu-repo/semantics/publishedVersio

Mining bioactive molecules from extreme environments using sequence-based metagenomics

The unique features of extreme habitats, particularly the high temperature and salinity, make them promising reservoirs for the bioprospection and discovery of novel and interesting bioactive molecules. In fact, the microorganisms inhabiting this type of environments have adopted survival strategies to thrive under hostile conditions, synthesizing several biochemical compounds valuable for many biotechnological applications. Culture-independent techniques, namely metagenomics, have emerged as powerful tools to access and explore the genetic and metabolic diversity of the microbial communities present in extreme and complex ecosystems. Two different metagenomic approaches can be applied, namely, sequence- and function-based metagenomics. The sequence-based studies allow finding new gene sequences that reveal similarities with the annotated genomes available in the databases. In this study, thermophilic composting samples were collected from two Portuguese composting units (Terra Fértil and Lipor), which handle different types of wastes. The metagenomic DNA was extracted from the composting samples and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to characterize the taxonomic and functional profiles of the metagenomes using both reads and metagenome-assembled genomes (MAGs) as input. Their microbiome was dominated by Bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria and Balneolia stood out for their higher abundance. These data confirm the richness of the composting habitats in bacterial strains known to survive and play an important role in harsh conditions, namely extreme saline environments. Furthermore, the combined analysis of functional annotation against KEGG and COG databases revealed a clear dominance of the contigs associated with the metabolism of carbohydrates, energy, amino acids and xenobiotics biodegradation and metabolism. This work represents the first comparative study on the taxonomic and functional profiles of Portuguese composting samples.info:eu-repo/semantics/publishedVersio

Functional screening for the detection of β-glucosidase activity in a metagenomic library obtained from a compost sample

There is an increasing need to find novel and robust biocatalysts with promising features that compete with those currently available on the market. Composting is an extreme habitat of high microbiological diversity that represents a suitable source of lignocellulose-degrading enzymes, such as cellulases, hemicellulases and ligninases, properly active under harsh conditions. These enzymes can convert the recalcitrant structure of lignocellulose into valuable bioproducts with great biotechnological potential. -Glucosidases are glycoside hydrolases responsible for degrading cellulose, namely in the disruption of the final glycosidic bonds of short-chain oligosaccharides to obtain glucose. Metagenomics is an emerging cultureindependent technique that has proven effective in identifying new biocatalysts with better catalytic activity through the analysis of DNA extracted from a vast number of environments. The metagenomic analysis is divided into two main technologies: sequence- and functionalbased approach. Function-based screening aims to discover and identify new genes capable of producing biocompounds/biomolecules with new or improved functions. This screening is based on the detection and isolation of clones with a positive response to the desired phenotype when activity-based techniques are applied. In this study, high-molecular-weight DNA extracted from a compost sample was used to construct a fosmid metagenomic library. This library was evaluated through a functional screening to identify clones that expressed cellulase activity, specifically -glucosidase activity. The enzymatic activity was unravelled using esculin as substrate through the formation of a brown halo as a positive response (Figure 1). The functional screening was performed in 96-well microplates and the detection of - glucosidase activity was evaluated at different temperature (25-60 °C) and pH (4.5-9.5) conditions. It was possible to identify clones with the enzymatic activity of -glucosidase in almost all tested conditions, except at 60 °C. The best conditions for clone growth occur in a longer initial incubation time (3 days, 37 °C). On the other hand, the lower pH and incubation temperature favoured a faster detection of -glucosidase activity.The study received financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIGNOZYMES (POCI-01-0145-FEDER-029773).info:eu-repo/semantics/publishedVersio

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

The online version contains supplementary material available at: https://doi.org/10.1007/s00253-023-12627-9.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 $\beta$-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.Open access funding provided by FCT|FCCN (b-on). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the projects LIGNOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773), and B3iS—Biodiversity and Bioprospecting of Biosurfactants in Saline Environments (PTDC/BII-BIO/5554/2020); and by RNCA Advanced Computing Project MetaLignoZymes, metagenomic analysis of lignocellulosic residues towards the discovery of novel enzymes (CPCA/A0/408464/2021).info:eu-repo/semantics/publishedVersio

Image processing tool for the detection and quantification of xylanase activity in a metagenomic study

The vast diversity of unexplored microbial communities inhabiting the planet drives the continuous screening for promising biocatalysts. Until recently, the strategies to find new microorganisms and their enzymes were mainly focused on laboratory studies of pure microbial cultures. However, a great amount of environmental microorganisms cannot be cultivated under laboratorial conditions [1]. Metagenomics has emerged as an innovative approach to explore these uncultivable microorganisms through the analysis of DNA extracted from environmental samples [2]. It is considered a powerful tool for the discovery of novel biocatalysts and two different approaches have been proposed. Sequence-based studies recognize candidate genes but do not provide direct conclusions about the functionality of the encoded enzymes. On the other hand, the function-based approach allows the identification of new enzymes and also leads to preliminary information about their activities and physicochemical parameters. Indeed, function-based screenings have been successfully used in different environments to find genes encoding lignocellulose-degrading enzymes, such as xylanases [2]. These enzymes are considered important catalysts in the biological decomposition of lignocellulosic residues. In this study, a fosmid library previously prepared in Escherichia coli with genomic DNA extracted from a compost sample collected in a national composting unit (Lipor) [3] was evaluated through a functional screening. To assess the xylanase activity of all the clones, a fast and simple chromogenic screening test using AZCL-xylan was performed in 96-well microplates at room temperature. Afterwards, the positive clones were selected and incubated at different temperatures (25, 37, 45 and 60 ºC) with the same substrate in Petri plates, for three days, to identify the most fast and promising clones. The presence of blue color was assumed as positive responses correlated with areas size. Area boundaries were extracted automatically by analyzing color images of the samples using MATLABs in-house functions. At 60 ºC, no positive clones were detected. Two positive clones simultaneously exhibited enzymatic activity under 25, 37 and 45 ºC. In general, 37 ºC proved to be the most suitable temperature for the detection of xylanase activity. The method herein reported can be further optimized for the automatic detection of different enzymatic activities in high throughput screenings.The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project LIGNOZYMES-Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01-0145-FEDER-029773). J.S. and A.M.A.C. acknowledge their research grants UMINHO/BIM/2020/28 and UMINHO/BPD/37/2018, respectively, under the scope of the project LIGNOZYMES. The authors also acknowledge the Portuguese composting unit Lipor for kindly supplying the compost sample.info:eu-repo/semantics/publishedVersio

Identification of lignocellulose-degrading enzymes using metagenomic approaches

Composting units which handle lignocellulosic residues are suitable sources of novel and promising lignocellulose-degrading enzymes such as cellulases, xylanases and amylases. These enzymes have practical application in many industries where lignocellulose is converted into several added-value bioproducts. However, the effective conversion of lignocellulose by a sustainable process is currently incomplete. Therefore, there is a need to find novel and robust catalysts to overcome this fact. Function- and sequence-based metagenomic approaches were used to identify novel lignocellulose-degrading enzymes with interesting industrial applications.info:eu-repo/semantics/publishedVersio

Hepatitis C Virus Infection as a Traumatic Experience

Objective The purpose of this study was to evaluate whether individuals consider their HCV infection to be a potentially traumatic experience. Additionally, we investigated its association with Post-Traumatic Stress Disorder (PTSD) and the impact of PTSD diagnosis on health-related quality of life (HRQoL) in HCV infected subjects. Methods We conducted a cross-sectional survey of 127 HCV-infected outpatients recruited at a University Hospital in Salvador, Brazil. All subjects answered an orally-administered questionnaire to gather clinical and socio-demographic data. We investigated traumatic experiences and the subject's perception of the disease using the Trauma History Questionnaire. PTSD and other psychiatric diagnoses were assessed through the Mini International Neuropsychiatric Interview-Brazilian Version 5.0.0 (M.I.N.I. PLUS). HRQoL was assessed using Short-Form 36 (SF-36). Results Approximately 38.6% of the patients considered hepatitis C to be a traumatic experience. Of these, 60.7% had a PTSD diagnosis. PTSD was associated with significant impairment in quality of life for individuals in seven SF-36 domains as shown bymultivariate analysis: Role-Physical (β: −24.85; 95% CI: −42.08; −7.61), Bodily Pain (β: −19.36; 95% CI: −31.28; −7.45), General Health (β: −20.79; 95% CI: −29.65; −11.92), Vitality (β: −11.92; 95% CI: −20.74; −3.1), Social Functioning (β: −34.73; 95% CI: −46.79; −22.68), Role-Emotional (β: −26.07; 95% CI: −44.61; −7.53), Mental Health (β: −17.46; 95% CI: −24.38; −10.54). Conclusion HCV is frequently a traumatic experience and it is strongly associated with PTSD diagnosis. PTSD significantly impaired HRQoL