Data-driven extraction of relative reasoning rules to limit combinatorial explosion in biodegradation pathway prediction

Motivation: The University of Minnesota Pathway Prediction System (UM-PPS) is a rule-based expert system to predict plausible biodegradation pathways for organic compounds. However, iterative application of these rules to generate biodegradation pathways leads to combinatorial explosion. We use data from known biotransformation pathways to rationally determine biotransformation priorities (relative reasoning rules) to limit this explosion. Results: A total of 112 relative reasoning rules were identified and implemented. In one prediction step, i.e. as per one generation predicted, the use of relative reasoning decreases the predicted biotransformations by over 25% for 50 compounds used to generate the rules and by about 15% for an external validation set of 47 xenobiotics, including pesticides, biocides and pharmaceuticals. The percentage of correctly predicted, experimentally known products remains at 75% when relative reasoning is used. The set of relative reasoning rules identified, therefore, effectively reduces the number of predicted transformation products without compromising the quality of the predictions. Availability: The UM-PPS server is freely available on the web to all users at the time of submission of this manuscript and will be available following publication at http://umbbd.msi.umn.edu/predict/. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

The University of Minnesota Biocatalysis/Biodegradation Database: improving public access

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.msi.umn.edu/) began in 1995 and now contains information on almost 1200 compounds, over 800 enzymes, almost 1300 reactions and almost 500 microorganism entries. Besides these data, it includes a Biochemical Periodic Table (UM-BPT) and a rule-based Pathway Prediction System (UM-PPS) (http://umbbd.msi.umn.edu/predict/) that predicts plausible pathways for microbial degradation of organic compounds. Currently, the UM-PPS contains 260 biotransformation rules derived from reactions found in the UM-BBD and scientific literature. Public access to UM-BBD data is increasing. UM-BBD compound data are now contributed to PubChem and ChemSpider, the public chemical databases. A new mirror website of the UM-BBD, UM-BPT and UM-PPS is being developed at ETH Zürich to improve speed and reliability of online access from anywhere in the world

PathPred: an enzyme-catalyzed metabolic pathway prediction server

The KEGG RPAIR database is a collection of biochemical structure transformation patterns, called RDM patterns, and chemical structure alignments of substrate-product pairs (reactant pairs) in all known enzyme-catalyzed reactions taken from the Enzyme Nomenclature and the KEGG PATHWAY database. Here, we present PathPred (http://www.genome.jp/tools/pathpred/), a web-based server to predict plausible pathways of muti-step reactions starting from a query compound, based on the local RDM pattern match and the global chemical structure alignment against the reactant pair library. In this server, we focus on predicting pathways for microbial biodegradation of environmental compounds and biosynthesis of plant secondary metabolites, which correspond to characteristic RDM patterns in 947 and 1397 reactant pairs, respectively. The server provides transformed compounds and reference transformation patterns in each predicted reaction, and displays all predicted multi-step reaction pathways in a tree-shaped graph

Data-driven extraction of relative reasoning rules to limit combinatorial explosion in biodegradation pathway prediction

Motivation: The University of Minnesota Pathway Prediction System (UM-PPS) is a rule-based expert system to predict plausible biodegradation pathways for organic compounds. However, iterative application of these rules to generate biodegradation pathways leads to combinatorial explosion. We use data from known biotransformation pathways to rationally determine biotransformation priorities (relative reasoning rules) to limit this explosion. Results: A total of 112 relative reasoning rules were identified and implemented. In one prediction step, i.e. as per one generation predicted, the use of relative reasoning decreases the predicted biotransformations by over 25% for 50 compounds used to generate the rules and by about 15% for an external validation set of 47 xenobiotics, including pesticides, biocides and pharmaceuticals. The percentage of correctly predicted, experimentally known products remains at 75% when relative reasoning is used. The set of relative reasoning rules identified, therefore, effectively reduces the number of predicted transformation products without compromising the quality of the predictions. Availability: The UM-PPS server is freely available on the web to all users at the time of submission of this manuscript and will be available following publication at http://umbbd.msi.umn.edu/predict/

Data-driven extraction of relative reasoning rules to limit combinatorial explosion in biodegradation pathway prediction

ISSN:1367-4803ISSN:1460-205

Molecular Similarity and Xenobiotic Metabolism

MetaPrint2D, a new software tool implementing a data-mining approach for predicting sites of xenobiotic metabolism has been developed. The algorithm is based on a statistical analysis of the occurrences of atom centred circular fingerprints in both substrates and metabolites. This approach has undergone extensive evaluation and been shown to be of comparable accuracy to current best-in-class tools, but is able to make much faster predictions, for the first time enabling chemists to explore the effects of structural modifications on a compound’s metabolism in a highly responsive and interactive manner.MetaPrint2D is able to assign a confidence score to the predictions it generates, based on the availability of relevant data and the degree to which a compound is modelled by the algorithm.In the course of the evaluation of MetaPrint2D a novel metric for assessing the performance of site of metabolism predictions has been introduced. This overcomes the bias introduced by molecule size and the number of sites of metabolism inherent to the most commonly reported metrics used to evaluate site of metabolism predictions.This data mining approach to site of metabolism prediction has been augmented by a set of reaction type definitions to produce MetaPrint2D-React, enabling prediction of the types of transformations a compound is likely to undergo and the metabolites that are formed. This approach has been evaluated against both historical data and metabolic schemes reported in a number of recently published studies. Results suggest that the ability of this method to predict metabolic transformations is highly dependent on the relevance of the training set data to the query compounds.MetaPrint2D has been released as an open source software library, and both MetaPrint2D and MetaPrint2D-React are available for chemists to use through the Unilever Centre for Molecular Science Informatics website.----Boehringer-Ingelhie

Development of metabolic pathways for the biosynthesis of hydroxyacids and lactones

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 178-210).In this thesis, metabolic routes were developed for the production of hydroxyacids and their lactones in multiple microbial systems. These compounds see widespread use in the production of pharmaceuticals, polymers, and fine chiral intermediates. First in this thesis, strategies and tools for metabolic pathway design are discussed. This is followed by the descriptions of and data for each microbial production system. The compounds produced in this thesis and their highest titers obtained are shown below: 3-Hydroxybutyrate (3HB) 2.9 g/L 3-Hydroxyvalerate (3HV) 5.3 g/L 4-Hydroxyvalerate (4HV) 27.1 g/L 4-Valerolactone (4VL) 8.2 g/L 3,4-Dihydroxybutyrate (DHBA) 3.2 g/L 3-Hydroxybutyrolactone (3-HBL) 2.2 g/L The production of the two hydroxyvalerates was accomplished through the reduction of the renewable substrate levulinate in Pseudomonas putida by endogenous host enzymes followed by the liberation of the hydroxyvalerate product through the recombinant expression of thioesterase B (tesB). The production of 4VL was accomplished from levulinate by adding the lactonase paraoxonase I (PON1) to the P. putida hydroxyvalerate production system. Because 4VL was found to exist in a pH-dependent equilibrium with 4HV, the lactonase was expressed extracytosolically in acidic media to achieve significant titers of 4VL. The addition of a second resin phase to 4VL-producing cultures with a high affinity for 4VL substantially enhanced lactone production. 3HB, 3HV, DHBA, and 3-HBL were all produced in Escherichia coli through the expression of an acetoacetyl-CoA thiolase (thil, bktB, or phaA), a 3-hydroxybutyryl-CoA reductase (phaB or hbd), and tesB.(cont.) Supplying glucose to E. coli expressing these enzymes resulted in 3HB production, while supplying glucose and propionate results in 3HV production. Supplying glucose and glycolate resulted in DHBA production with some 3-HBL, but only with the help of a fourth gene - propionyl-CoA transferase (pct). Removing the tesB gene from this four-gene system substantially increases 3-HBL titers at the expense of DHBA. This work represents the first successful production of DHBA and 3-HBL in a biological system from carbohydrate-based substrates. In each of these systems, several broadly-applicable tools and strategies were developed to enhance product titer or discover new metabolic activities. In the P. putida system, cytosolic and extracytosolic biocatalysis were combined in a single metabolic pathway to realize lactone production. This catalytic strategy, termed integrated bioprocessing, is applicable to other metabolic pathways whose production suffers due to a suboptimal cytosolic enzyme. Also in the P. putida system, two-phase cultures were used to sequester the lactone product away from the lactonase, helping to drive lactonehydroxyacid equilibrium towards the lactone. This methodology allows one to overcome equilibrium-based limitations of product titer. Finally in the E. coli work, a promiscuous pathway normally used for polyhydroxyalkanoate synthesis was exploited to give a wide range of hydroxyacid products. This substrate promiscuity was critical in achieving the production of new compounds biologically and thus substrate promiscuity was identified as a key component for metabolic pathway design and construction.by Collin H. Martin.Ph.D

Identification of transformation products of emerging contaminants during tertiary treatment processes and their disposal in the environment by mass spectrometric techniques

Η διαθέσιμη βιβλιογραφία έχει καταδείξει την ατελή απομάκρυνση των αναδυόμενων ρύπων κατά τη βιολογική επεξεργασία που λαμβάνει χώρα στα Κέντρα Επεξεργασίας Λυμάτων (ΚΕΛ). Παρόλο που ο πρωταρχικός ρόλος της τριτοβάθμιας επεξεργασίας είναι η εξάλειψη των μικρορύπων, το παρεχόμενο οξειδωτικό μέσο αντιδρά με τους αναδυόμενους ρύπους, καταλήγοντας στο σχηματισμό άγνωστων προϊόντων μετασχηματισμού. Ο κύριος στόχος της παρούσας διπλωματικής εργασίας ήταν η διερεύνηση της απομάκρυνσης αναδυόμενων ρύπων καθώς και η ταυτοποίηση των προϊόντων μετασχηματισμού τους, τα οποία παράγονται κατά τις μεθόδους απολύμανσης που εφαρμόζονται σαν τριτοβάθμια επεξεργασία στα ΚΕΛ. Αρχικά παρουσιάζεται μια εισαγωγή για τους αναδυόμενους ρύπους και τον πιθανό μετασχηματισμό τους κατά τη διάρκεια των διεργασιών που εφαρμόζονται στα ΚΕΛ, και ιδιαιτέρως κατά τη διάρκεια της χλωρίωσης και οζόνωσης. Στη συνέχεια παρουσιάζονται συγκεκριμένες πορείες εργασίας και τεχνικές σχετικές με την ταυτοποίηση αναδυόμενων ρύπων και των προϊόντων μετασχηματισμού τους, οι οποίες βασίζονται σε αναλύσεις φασματομετρίας μάζας υψηλής διακριτικής ικανότητας. Το πειραματικό μέρος της παρούσας διπλωματικής εργασίας αποτελείται από τα ακόλουθα τρία μέρη: (α) Χλωρίωση βενζοτριαζολών και βενζοθειαζολών και ταυτοποίηση των προϊόντων μετασχηματισμού τους με υγροχρωματογραφία συζευγμένη με φασματομετρία μάζας υψηλής διακριτικής ικανότητας (Κεφάλαιο 3), (β) Οζόνωση ρανιτιδίνης: επίδραση των πειραματικών συνθηκών και ταυτοποίηση προϊόντων μετασχηματισμού (Κεφάλαιο 4) και (γ) Απομάκρυνση και μετασχηματισμός της σιταλοπράμης και τεσσάρων προϊόντων βιομετατροπής της κατά τη διάρκεια πειραμάτων οζόνωσης (Κεφάλαιο 5). Είναι πεποίθησή μας πως οι μελέτες αυτές θα συνεισφέρουν στην περαιτέρω ανάπτυξη της περιβαλλοντικής ανάλυσης, διεγείροντας το ενδιαφέρον των κανονιστικών αρχών, σχετικά με την ενσωμάτωση επιβλαβών προϊόντων μετασχηματισμού, τα οποία παράγονται κατά την τριτοβάθμια επεξεργασία των ΚΕΛ, σε ελέγχους ρουτίνας.The incomplete removal of emerging pollutants during the biological treatment applied in WWTPs is highly indicated by the existing literature. Although the primary purpose of tertiary treatment processes is the elimination of micropollutants, the provided oxidant agent reacts with emerging pollutants, leading to the formation of unknown transformation products. The main objectives of this thesis were the investigation of the removal of emerging pollutants and the identification of their transformation products which are produced during the disinfection methods that are applied as tertiary treatment is WWTPs. Initially, an introduction on emerging pollutants and their probable transformation during the processes that are applied in WWTPs, and especially during chlorination and ozonation, is presented. Specific workflows and techniques for the identification of emerging contaminants and their transformation products, based on high-resolution mass spectrometric analysis, are then presented. The experimental section of this thesis is constituted of the following three parts: (i) Chlorination of benzothiazoles and benzotriazoles and transformation products identification by LC-HR-MS/MS (Chapter 3), (ii) Ozonation of ranitidine: effect of experimental parameters and identification of transformation products (Chapter 4) and (iii) Removal and transformation of citalopram and four of its biotransformation products during ozonation experiments (Chapter 5). It is our strong belief that these studies will constitute a step forward in environmental analysis, by arousing the regulatory authorities concern, regarding that harmful transformation products produced during tertiary treatment processes in WWTPs, should also be incorporated in routine monitoring

Development of Mass Spectrometric Methods for the determination of Emerging Pollutants in wastewater

Η παρουσία αναδυόμενων ρύπων σε περιβαλλοντικά δείγματα είναι αδιαμφισβήτητη, καθώς αστικές, βιομηχανικές και γεωργικές δραστηριότητες αποβάλλουν στο υδάτινο περιβάλλον μεγάλες ποσότητες διαφόρων οργανικών μικρορυπαντών, τα τελευταία χρόνια. Η παρακολούθηση και ο έλεγχος του περιβάλλοντος αποτελεί ανάγκη στις μέρες μας, σαν μέρος μιας συνολικής περιβαλλοντικής αξιολόγησης κινδύνου. Ο κύριος στόχος της παρούσας διπλωματικής διατριβής ήταν η ανάπτυξη ευαίσθητων αναλυτικών μεθοδων για το προσδιορισμό αναδυόμενων ρύπων από διάφορες κατηγορίες, με τη χρήση διαφορετικών τεχνικών φασματομετρίας μάζας. Αρχικά παρουσιάζεται μια εκτεταμένη ανασκόπηση σχετικά με τους αναδυόμενους ρύπους, καθώς περικλείουν διάφορες ομάδες ενώσεων, τονίζοντας το εύρος των ιδιοτήτες τους. Συγκεκριμένες πορείες και τεχνικές παρουσιάζονται, στη συνέχεια, σχετικά με τον προσδιορισμό τους, στοχεύοντας κυρίως στη στοχευμένη ανάλυση και στη παρακολούθηση “’ύποπτων” ενώσεων, με τεχνικές φασματομετρίας μάζας και κυρίως στην ανάπτυξη της φασματομετρίας μάζας υψηλής διακριτικής ικανότητας. Το πειραματικό μέρος της διατριβής αποτελείται από τρία μέρη: (α) Προσδιορισμός σιλοξανίων με αέριοχρωματογραφια συζευγμένης με φασματομετρίας μαζών και εκτίμηση του φορτίου και της τύχης τους σε ένα κέντρο επεξεργασίας λυμάτων σην Ελλάδα (Κεφάλαιο 3), (β) Ποσοτική στοχευμένη ανάλυση 2327 αναδυόμενων ρύπων σε λύματα με υγροχρωματογραφία υψηλής απόδοσης συζευγμένης με φασματομετρία μάζας τεχνολογίας τετραπόλου-αναλυτή χρόνου πτήσης (Κεφάλαιο 4) και (γ) Παρακολούθηση “’ύποπτων” ενώσεων για το χαρακτηρισμό πολικών οργανικών αναδυόμενων ρύπων σε λύματα με υγροχρωματογραφία υψηλής απόδοσης συζευγμένης με φασματομετρία μάζας υψηλής διακριτικής ικανότητας. Οι μελέτες αυτές αποτελούν ένα σημαντικό βήμα στην ανάλυση περιβαλλοντικών αναδυόμενων ρύπων.The presence of emerging pollutants in environmental samples is unquestionable, since urban, industrial and agricultural activities release in the aquatic environment great amounts of diverse organic micropollutants over the last decade. Environmental monitoring is an urgent nowadays, as a part of a holistic environmental risk assessment. The main objective of this thesis was the development of highly sensitive methods for the determination of multi-class emerging pollutants with the use of different mass spectrometric techniques. First, an introduction on the emerging pollutants is presented, since they encompass a diverse group of compounds, highlighting their wide-scope properties. Specific workflows and techniques for their determination are then presented, focusing in target and suspect screening by mass spectrometric techniques and especially in the development of high-resolution mass analyzers. The experimental section of the thesis consists of three parts: (i) Determination of Linear and Cyclic Siloxanes by gas chromatography hyphenated with mass spectrometry in environmental samples and evaluation of Mass Loadings and Fate in a Wastewater Treatment Plant in Greece (Chapter 3), (ii) Wide-scope quantitative target screening of 2327 emerging contaminants in wastewater samples with Ultra-Performance Liquid Chromatography Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-ToF-HRMS/MS) (Chapter 4) and (iii) Suspect screening workflow for the characterization of emerging polar organic contaminants in wastewater samples with Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS/MS) (Chapter 5). It is our strong belief that these studies will constitute a step forward in environmental analysis of emerging pollutants

Biodegradation of the steroid progesterone in surface waters

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonMany studies measuring the occurrence of pharmaceuticals, understanding their environmental fate and the risk they pose to surface water resources have been published. However, very little is known about the relevant transformation products which result from the wide range of biotic and abiotic degradation processes that these compounds undergo in sewers, storage tanks, during engineered treatment and in the environment. Thus, the present study primarily investigated the degradation of the steroid progesterone (P4) in natural systems (rivers), with a focus on the identification and characterisation of transformation products. Initial work focussed on assessing the removal of selected compounds (Diclofenac, Fluoxetine, Propranolol and P4) from reed beds, with identification of transformation products in a field site being attempted. However, it was determined that concentrations of parent compounds and products would be too low to work with in the field, and a laboratory study was designed which focussed on P4. Focus on P4 was based on literature evidence of its rapid biodegradability relative to the other model compounds and its usage patterns globally. River water sampling for the laboratory-based degradation study was carried out at 1 km downstream of four south east England sewage works (Blackbirds, Chesham, High Wycombe and Maple Lodge) effluent discharge points. Suspected P4 transformation products were initially identified from predictions by the EAWAG Biocatalysis Biodegradation Database (EAWAG BBD) and from a literature review. At a later stage of the present work, a replacement model for EAWAG BBD (enviPath) which became available, was used to predict P4 degradation and results were compared. Samples were analysed using low resolution and accurate-mass time-of-flight mass spectrometers. Three degradation studies were conducted. Sampling for all studies was carried out at the same time in the year to minimize temporal variability in conditions and allow for effective comparison of results. Androgenic and progesterone yeast screens were carried out to assess the biological activity of transformation products.Nigeria Tertiary Education Trust Fund (TETFund), the Uduaghan led-administration of the Delta State Government, the 2010-2015 board members of the Delta State Scholarship Board