INVESTIGATIONS ON A CHLOROFORM REDUCTIVE DEHALOGENASE Bat-Erdene Jugder A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science July 2017 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: JUGDER First name: BAT-ERDENE Other name/s: Abbreviation for degree as given in the University calendar: PhD School: School of Biotechnology and Biomolecular Sciences Faculty: Science Title: Investigations on a chloroform reductive dehalogenase Abstract 350 words maximum: (PLEASE TYPE) Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic and carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide respiring bacteria provide a potential solution to remediate contaminated sites, as they are capable of utilising organohalides as electron acceptors for the generation of cellular energy. At the heart of these processes are reductive dehalogenases (RDase; EC 1.97.1.8), which are membrane bound enzymes that catalyse reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Chloroform (CF), primarily used in the production of refrigerants, is very prevalent and recalcitrant organohalide contamination and its improper disposal also caused groundwater pollution at industrial sites in Sydney, Australia. Its hazardous effect on environment and carcinogenic and organo-toxic effects on human health prompts bioremediation research towards its detoxification. Recently, Dehalobacter (Dhb) sp. strain UNSWDHB, which is capable of respiring CF and converting it to dichloromethane has been identified. The UNSWDHB strain was revealed to produce CF-RDase, as termed TmrA, which has been the focus of the research carried out in this thesis. Firstly, the response of Dhb sp. UNSWDHB to the addition of CF was evaluated from a transcriptomic and proteomic perspective. The elevated expressions of TmrABC proteins, key bioenergetics related membrane-associated and cytoplasmic proteins, enzymes associated with functional Wood-Ljungdahl pathway and complete corrinoid de novo synthesis were revealed, providing a broader view on the bioenergetics and general physiology of the Dehalobacter cells actively respiring with CF. Furthermore, TmrA was produced and purified from the membrane fraction of the UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and anion exchange chromatographic purification. This allowed further biochemical characterisation of the purified enzyme. Lastly, an extensive study to heterologously express TmrA was conducted under several expression conditions to address a reportedly challenging issue with soluble, functional expression and purification of a respiratory RDase. The xylose-inducible expression in a corrinoid-producing Bacillus megaterium as an expression host and two liquid chromatographic steps resulted in a generation of a soluble and functional recombinant TmrA. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied//wwill apply for a parti al restriction of the digital copy of my thesis or dissertation.' Signed ………………………………………………………………………………………............. Date ………………………10 July 2017…………………………………………………............. AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………………………………………………….............. Date ………………………10 July 2017…………………………………………………............. iv ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………10 July 2017…………………….............. iii Abstract Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic and carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide respiring bacteria provide a potential solution to remediate contaminated sites, as they are capable of utilising organohalides as electron acceptors for the generation of cellular energy. At the heart of these processes are reductive dehalogenases (RDase; EC 1.97.1.8), which are membrane bound enzymes that catalyse reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Chloroform (CF), primarily used in the production of refrigerants, is very prevalent and recalcitrant organohalide contamination and its improper disposal also caused groundwater pollution at industrial sites in Sydney, Australia. Its hazardous effect on environment and carcinogenic and organo-toxic effects on human health prompts bioremediation research towards its detoxification. Recently, Dehalobacter (Dhb) sp. strain UNSWDHB, which is capable of respiring CF and converting it to dichloromethane has been identified. The UNSWDHB strain was revealed to produce CF-RDase, as termed TmrA, which has been the focus of the research carried out in this thesis. Firstly, the response of Dhb sp. UNSWDHB to the addition of CF was evaluated from a transcriptomic and proteomic perspective. The elevated expressions of TmrABC proteins, key bioenergetics related membrane-associated and cytoplasmic proteins, enzymes associated with functional Wood-Ljungdahl pathway and complete corrinoid de novo synthesis were revealed, providing a broader view on the bioenergetics and general physiology of the Dehalobacter cells actively respiring with CF. Furthermore, TmrA was produced and purified from the membrane fraction of the UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and anion exchange chromatographic purification. This allowed further biochemical characterisation of the purified enzyme. Lastly, an extensive study to heterologously express TmrA was conducted under several expression conditions to address a reportedly challenging issue with soluble, functional expression and purification of a respiratory RDase. The xylose-inducible expression in a corrinoid-producing Bacillus i megaterium as an expression host and two liquid chromatographic steps resulted in a generation of a soluble and functional recombinant TmrA. ii Acknowledgement There are many individuals whom I would like to express my gratitude and appreciation for their help in my pursuit of a PhD at UNSW Sydney. The first and foremost thanks must go out to my family: my parents, wife, son and daughter. I would not be writing this acknowledgement if I were not fortunate enough to have my wife and best friend Alta in my life, whose love and encouragement are always beyond imagination and expectation.