ANASTASIA: An Automated Metagenomic Analysis Pipeline for Novel Enzyme Discovery Exploiting Next Generation Sequencing Data

Metagenomic analysis of environmental samples provides deep insight into the enzymatic mixture of the corresponding niches, capable of revealing peptide sequences with novel functional properties exploiting the high performance of next-generation sequencing (NGS) technologies. At the same time due to their ever increasing complexity, there is a compelling need for ever larger computational configurations to ensure proper bioinformatic analysis, and fine annotation. With the aiming to address the challenges of such an endeavor, we have developed a novel web-based application named ANASTASIA (automated nucleotide aminoacid sequences translational plAtform for systemic interpretation and analysis). ANASTASIA provides a rich environment of bioinformatic tools, either publicly available or novel, proprietary algorithms, integrated within numerous automated algorithmic workflows, and which enables versatile data processing tasks for (meta)genomic sequence datasets. ANASTASIA was initially developed in the framework of the European FP7 project HotZyme, whose aim was to perform exhaustive analysis of metagenomes derived from thermal springs around the globe and to discover new enzymes of industrial interest. ANASTASIA has evolved to become a stable and extensible environment for diversified, metagenomic, functional analyses for a range of applications overarching industrial biotechnology to biomedicine, within the frames of the ELIXIR-GR project. As a showcase, we report the successful in silico mining of a novel thermostable esterase termed “EstDZ4” from a metagenomic sample collected from a hot spring located in Krisuvik, Iceland

Using Prokaryotes for Carbon Capture Storage

Geological storage of CO2 is a fast-developing technology that can mitigate rising carbon emissions. However, there are environmental concerns with long-term storage and implications of a leak from a carbon capture storage (CCS) site. Traditional monitoring lacks clear protocols and relies heavily on physical methods. Here we discuss the potential of biotechnology, focusing on microbes with a natural ability to utilize and assimilate CO2 through different metabolic pathways. We propose the use of natural microbial communities for CCS monitoring and CO2 utilization, and, with examples, demonstrate how synthetic biology may maximize CO2 uptake within and above storage sites. An integrated physical and biological approach, combined with metagenomics data and biotechnological advances, will enhance CO2 sequestration and prevent large-scale leakages

Discovery and characterization of a thermostable and highly halotolerant GH5 cellulase from an Icelandic hot spring isolate

Journal ArticleCopyright: © 2016 Zarafeta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.This work has been carried out in the framework of the HotZyme Project (http://hotzyme.com, grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013, an EU FP7 Collaborative programme

Metagenomic analysis for the detection of novel microbial enzymes of biotechnological interest

Aim of this thesis was the development of an automated bioinformatic framework that could effectively handle and analyze metagenomic data with the final scope being the detection of novel enzymes of industrial interest. The design of the aforementioned framework comprised evaluating various open source bioinformatic tools via running multiple analyses in real metagenomic datasets, as well as developing new algorithms that could tackle any issues derived from these analyses. The selected tools and developed algorithms were integrated in a web-based platform which was developed by exploiting Galaxy's computational framework and was named ANASTASIA (Automated Nucleotide Aminoacid Sequences Translational plAtform for Systemic Interpretation and Analysis). This new platform offered a friendly graphic user interface for all tools incorporated in it, while enabling the automation of each analysis in which they were executed, through the use of appropriate computational pipelines. The design of the computational pipelines was facilitated by using the integrated tools directly on real metagenomic datasets acquired from two research projects: HotZyme and COVERALL. HotZyme project aimed in discovering novel thermostable enzymes via metagenomic screening of environmental samples from terrestrial hot springs, while COVERALL focused on detecting the taxonomical and functional differences in metagenomic samples from seafloor sediments, that were exposed in high concentrations of CO2. The bioinformatic analysis in both projects was initiated at the level of metagenomic sequencing data, but was formed in two distinct methodologies of different analytical steps, which were later transformed into the corresponding automated bioinformatic pipelines. The final version of the platform, including the automated pipelines and the integrated tools they consist of, was rendered available online, by using a server owned by the school of Chemical Engineering in National Technical University of Athens, via the following URL: http://motherbox.chemeng.ntua.gr/anastasia_dev/.The results from HotZyme project consisted of a list of nucleotide sequences of putative hydrolytic activity (EC number: 3.-.-.-) which was further curated in order to select the most promising candidates for experimental validation. During the work for this thesis, two of those sequences were successfully isolated in the lab, expressed, fully annotated and registered in public databases (UniProt) as novel sequences, while confirming our initial prediction regarding their enzymatic activity. On COVERALL project, the utilization of ANASTASIA resulted in identifying 23 different species that were found present only during exposure to high concentrations of CO2. The corresponding genomic sequences were parsed and are already under way of further analysis for detecting distinctive biomarker sequences for exposure in that pollutant. Designing this platform through the constant interaction of real metagenomic data, was of utmost importance for evaluating its potential, as well as for readjusting its algorithms for further optimizing the handling and analyzing of the corresponding files. Thus, the significant outcome of this thesis does not consist solely of the analysis results for each project but also of the user friendly and constantly evolving computational framework that was developed. The potential of that framework has already been proven in the case of metagenomic data but can be further (and already is) expanding for all fields of Biotechnology, -omics technologies and Synthetic Biology.Σκοπός της παρούσας διατριβής ήταν η ανάπτυξη ενός αυτοματοποιημένου συστήματος βιοπληροφορικών αναλύσεων, το οποίο θα μπορούσε να διαχειριστεί και να αναλύσει μεταγενωμικά δεδομένα, με τελικό στόχο την εύρεση καινούριων ενζύμων βιοτεχνολογικού ενδιαφέροντος. Η διαδικασία σχεδιασμού του αυτοματοποιημένου συστήματος περιελάμβανε την αξιολόγηση πολυάριθμων βιοπληροφορικών εργαλείων μέσω της εφαρμογής τους σε πραγματικά μεταγενωμικά δεδομένα καθώς και την ανάπτυξη καινούριων αλγορίθμων που καλύπτουν τις αδυναμίες των ήδη υπαρχόντων. Η συλλογή των διαφορετικών εργαλείων και των νέων αλγορίθμων ενοποιήθηκε σε μία διαδικτυακή πλατφόρμα που κατασκευάστηκε με βάση το υπολογιστικό σύστημα Galaxy και ονομάστηκε ANASTASIA (Automated Nucleotide Aminoacid Sequences Translational plAtform for Systemic Interpretation and Analysis). Στην καινούρια πλατφόρμα το κάθε εργαλείο και αλγόριθμος γινόταν διαθέσιμο μέσω ενός φιλικού προς το χρήστη γραφικού περιβάλλοντος ενώ υπήρχε η δυνατότητα αυτοματοποίησης των αναλύσεων που περιλάμβαναν πολλά διαδοχικά εργαλεία μέσα από βιοπληροφορικές γραμμές εργασιών (pipelines). Ο σχεδιασμός των αυτοματοποιημένων γραμμών εργασιών έγινε μέσω της εφαρμογής των ενσωματωμένων εργαλείων σε μεταγενωμικά δεδομένα που αποκτήθηκαν από δύο ερευνητικά προγράμματα: το HotZyme και το COVERALL. Το πρώτο πρόγραμμα είχε ως σκοπό την εύρεση καινούριων θερμοσταθερών ενζύμων μέσω ανάλυσης των μεταγονιδιωμάτων μικροβιακών πληθυσμών σε θερμές πηγές, ενώ το δεύτερο πραγματευόταν την εύρεση μικροβιακών ειδών και αλληλουχιών που θα μπορούσαν να συσχετιστούν ως βιοδείκτες με την έκθεση σε υψηλές συγκεντρώσεις CO2. Η βιοπληροφορική ανάλυση και στα δύο ερευνητικά προγράμματα ξεκίνησε από το επίπεδο των δεδομένων αλληλούχισης των μεταγονιδιωμάτων των αντίστοιχων δειγμάτων αλλά εξελίχθηκε σε δύο διαφορετικές μεθοδολογίες από τις οποίες προέκυψαν οι αντίστοιχες αυτοματοποιημένες γραμμές εργασιών. Η τελική έκδοση της πλατφόρμας με τα ενσωματωμένα εργαλεία και τις αντίστοιχες αυτοματοποιημένες γραμμές εργασιών έγινε στη συνέχεια διαθέσιμη διαδικτυακά αξιοποιώντας ένα διακομιστή που ανήκει στη σχολή Χημικών Μηχανικών ΕΜΠ στη διεύθυνση http://motherbox.chemeng.ntua.gr/anastasia_dev/.Από τα αποτελέσματα της ανάλυσης των δεδομένων του ερευνητικού προγράμματος HotZyme προέκυψε μία λίστα αλληλουχιών πιθανών ενζύμων με υδρολυτική δράση (αριθμός EC 3.-.-.-) η οποία εξετάστηκε περαιτέρω για την επιλογή των επικρατέστερων υποψηφίων για εργαστηριακή επιβεβαίωση. Κατά τη συγγραφή αυτής της διατριβής ήδη δύο από τις παραπάνω αλληλουχίες έχουν απομονωθεί στο εργαστήριο, έχουν εκφραστεί επιτυχώς, έχουν χαρακτηριστεί πλήρως ως προς την ενζυμική λειτουργία τους και έχουν καταγραφεί σε δημόσιες βάσεις δεδομένων (UniProt) ως καινούριες καταχωρήσεις, επιβεβαιώνοντας τις αρχικές μας προβλέψεις. Αντίστοιχα τα αποτελέσματα του ερευνητικού προγράμματος COVERALL αποκάλυψαν 23 διαφορετικά μικροβιακά είδη των οποίων η παρουσία φαίνεται να συνδέεται στενά με την έκθεση σε υψηλές συγκεντρώσεις CO2. Οι αντίστοιχες γονιδιακές τους αλληλουχίες έχουν ήδη απομονωθεί υπολογιστικά και ήδη λαμβάνει χώρα περαιτέρω ανάλυση για τον εντοπισμό χαρακτηριστικών αλληλουχιών που θα αποτελέσουν πιθανούς βιοδείκτες έκθεσης για το συγκεκριμένο ρύπο. Ο σχεδιασμός αυτής της πλατφόρμας, μέσα από τη συνεχή αλληλεπίδραση με πραγματικά μεταγενωμικά δεδομένα, βοήθησε εξαιρετικά στην αξιολόγηση των δυνατοτήτων της, αλλά και στην αναπροσαρμογή των αλγορίθμων της για τη βέλτιστη διαχείριση και ανάλυση των αντίστοιχων αρχείων. Έτσι το ουσιαστικό αποτέλεσμα αυτής της διατριβής δεν αποτελείται μόνο από τα συμπεράσματα των εκάστοτε αναλύσεων, αλλά σαφώς επίσης και από το εύχρηστο και διαρκώς εξελισσόμενο υπολογιστικό σύστημα που προέκυψε. Οι δυνατότητες αυτού του συστήματος ενώ έχουν αποδειχτεί για την περίπτωση μεταγονιδιωματικών δεδομένων μπορούν να επεκταθούν (και ήδη επεκτείνονται) περαιτέρω για όλους τους τομείς της Βιοτεχνολογίας και της Συνθετικής Βιολογίας

Thermostability of CelDZ1.

<p><b>(A).</b> Catalytic thermostability of CelDZ1 evaluated by measurements of residual CMC-degrading activity after high-temperature exposure at 65, 70 and 75°C for up to 24 h. <b>(B).</b> Thermal denaturation analysis of CelDZ1 using differential scanning fluorimetry with the conformation-sensitive dye SYPRO Orange. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.</p

Halostability and halotolerance of CelDZ1.

<p><b>(A)</b> CelDZ1 was incubated in 5 M NaCl and 4 M KCl for up to 20 days. At different time intervals aliquots were taken and the residual activity of the enzyme was measured in the standard reaction. <b>(B)</b> The activity of CelDZ1 in the presence of different high-salt concentrations was measured in the standard reaction. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.</p

Effect of pH and temperature on the activity of CelDZ1.

<p><b>(A)</b> CelDZ1 activity was measured in the standard reaction at 40°C for 5 min at pH values ranging from 4 to 10 and <b>(B)</b> at temperatures between 40 and 90°C for 5 min in a pH 5 buffer. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.</p

The structure of CelDZ1.

<p><b>(A)</b> Folding of the CelDZ1α monomer is presented as a cartoon diagram and viewed from the solvent region towards the active site groove formed by the C-terminal ends of the β-strands of the (β/α)₈-barrel. The α-helices, β-strands and loops are coloured in turquoise, magenta and pink, respectively. The carbohydrate-binding module (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.g001" target="_blank">Fig 1</a>), which contains helix α8 at the C terminus, is highlighted in green. The two catalytic residues are shown as stick models and secondary structural elements are labelled. The Met50 indicates the position of the first N-terminal residue defined in the electron density. The image was prepared using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref040" target="_blank">40</a>]. <b>(B)</b> A stereo representation of the superimposition of the monomers of CelDZ1, CelK and Cel5a displayed as grey carbon traces. The three different insertion regions are highlighted in red for CelDZ1, magenta for CelK and green for Cel5a. The cellobiose ligand bound to CelK is shown as a magenta stick model. The image was prepared using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref040" target="_blank">40</a>]. <b>(C)</b> The electrostatic potential surface of the CelDZ1 enzyme around the active site groove as viewed from the solvent region. The positive charge is shown in blue and the negative charge is shown in red. The extended active site groove, which crosses the monomer from left to right, is negatively charged disfavoring the binding of halogen ions thereby increasing halotolerance. The image was prepared with ccp4mg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref041" target="_blank">41</a>].</p

Discovery of the cellulolytic enzyme CelDZ1.

<p><b>(A)</b> The amino acid sequence of the putative cellulolytic enzyme corresponding to the <i>celDZ1α</i> ORF was analysed against the Pfam-A database. The analysis revealead that the predicted sequence consists of a GH5 catalytic domain (denoted as domain 1), a carbohydrate-binding module (CBM) 17/28 (denoted as domain 2) and a transmembrane anchor (denoted as domain 3). <b>(B)</b> Zymogram analysis for the detection of cellulolytic activity via SDS-PAGE analysis and Congo red staining of a CMC-containing acrylamide gel. M: molecular weight marker; 1: cell lysate producing the target protein 2: cell lysate carrying an empty vector.</p