Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

BACKGROUND: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. RESULTS: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g(-1) dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg(-1) dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g(-1) dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. CONCLUSIONS: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Study of marine oil polluted marine sites by the ULIXES, MAGICPAH and KILLSPILL consortia

Trabajo presentado en el 10th International Congress on Extremophiles, celebrado en San Petesburgo (Rusia) del 07 al 11 de septiembre de 2014.Oil polluted sites are another example of extreme enviroments where the changes in the media due to contamination cause a shift in the microbial distribution, rising the presence of those species able to survive in these conditions and also with capabilities to catch and metabolize the different oil compounds like linear and branched alkanes and polyaromatic hydrocarbons (PAHs), which are very recalcitrant substances that contribute to the ecological alteration of the enviroment. ULIXES, MAGICPAH and KILLSPILL are EU-funded projects focus on cataloguing and targeting, by using state-of-the-art technologies, how this type of pollution influence microbial diversity and ecology, and developing novel biorremediation methods based on novel knowledge from the results obtained by researching polluted sites. Some of the efforts in this investigation are focused in use computational techniques to identify functional groups related with degradation of pollutants generated by oil spills, providing an easier prediction of the system behaviour and the experimental analysis design. Here we describe the computational methods used to reach this goal, based on the study different oil pulluted sites across the Northern and Southern Mediterranean area and the Gulf of Aqaba (Jordan) at Red Sea.Peer Reviewe

The ULIXES, MAGICPAH and KILLSPILL Projects: methodology and initial computational results

Trabajo presentado en la International Conference (MedRem) celebrada en Hammamet (Túnez) del 16 al 18 de enero de 2014.ULIXES, MAGICPAH and KILLSPILL are EU-funded projects that are focused in (i) cataloguing and targeting, by using state-of-the-art technologies, primarily, the way pollution with oil hydrocarbons and/or polyaromatic hydrocarbons influence microbial diversity and ecology and, (ii) secondly, developing novel bioremediation process based on novel knowledge accumulated by investigating marine (ULIXES and KILLSPILL) and soil (MAGICPAH) polluted sites. The projects comprises several interrelated efforts, one being the identification of functional groups by using computational strategies, which may ultimately serve as the basis for making a comprehensive repository of function (mainly the one involved in biodegradation) prediction and experimental analysis. Here we provide some features about the overall design, the computational resources, and draft results from the initial and last phases of the projects, focused in polluted site across sites at the Northern and Southern Mediterranean side as well as the Gulf of Aqaba (Jordan) at Red Sea.Peer Reviewe

Dissertationis academicae particula prior, de claritate pugnae Themistocleae apud Salaminam, quam ... praeside ... mag. Petro Ekerman ... examen, Jacobus M. Hallgren, Gevalia-Gestriicus. In aud. Carol. maj. ad diem [] Aprilis, anni MDCCLXII. Horis ante merid. consvetis.

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials

Functional enzyme diversity in a number of extreme and non-extreme environments

Trabajo presentado en el 10th International Congress on Extremophiles, celebrado en San Petesburgo (Rusia) del 07 al 11 de septiembre de 2014.The collaborative projects, MAMBA, MAGICPAH, ULIXES, funded by the EU FP7 program, and the BIO2011- 25012 (funded by Spanish Ministry of Economy and Competitiveness) are focused on using environmental sites around the Planet (including soils, acid mines, superficial and deep seawater, etc.) as resources of novel microbial assemblages, gene sequences and enzyme activities, some of which could have potential scientific and industrial applications. Some of the environments, that included contaminated and concontaminated sites, may be inhabited by extremophiles that contains “extremozymes”. The investigation of their “extremozymes”, and their new functions and protein folds, and the correlation between those characteristics and environmental constraints is of interest for achieving the full understating of extreme ecosystem functioning. Here, using cultivation-independent techniques, and in particular, metagenomics, we have selected and analyzed microbial communities from various extreme and non extreme environments and provided a draft vision into their enzyme properties.The authors gratefully acknowledge the financial support provided by the European Community projects ULIXES (FP7-KBBE-2010-266473), MAGICPAH (FP7-KBBE-2009-245226), and MAMBA (FP7-KBBE-2008-226977). This work was further funded by grant BIO2011-25012 from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation

María Eugenia Guazzaroni et al.Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted OMIC investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH 4 NO 3 and KH 2 PO 4 and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct presumptive degradation networks for complex microbial communities. © 2013 International Society for Microbial Ecology All rights reserved.This research was supported by the Spanish CSD2007-00005 and CENIT-07-CLEAM projects and by FEDER funds, ERANET (GEN2006-27750-C5-5-E/SYS), and the European Community Projects MAGICPAH (FP7-KBBE-2009-245226) and ULIXES (FP7-KBBE-2010-266473). M-EG thanks the CSIC for a JAE fellowship, and IL thanks FICYT, Principado de Asturias for a Severo Ochoa fellowshipPeer Reviewe

Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

Abstract Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases