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Essential Cofactors in Anaerobic Microbial Consortia Used for Bioremediation: Biosynthesis, Function and Regeneration

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Essential Cofactors in Anaerobic Microbial Consortia Used for Bioremediation: Biosynthesis, Function and Regeneration ESSENTIAL COFACTORS IN ANAEROBIC MICROBIAL CONSORTIA USED FOR BIOREMEDIATION: BIOSYNTHESIS, FUNCTION AND REGENERATION by Po-Hsiang Wang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemical Engineering and Applied Chemistry University of Toronto © Copyright by Po-Hsiang Wang (2018) ESSENTIAL COFACTORS IN ANAEROBIC MICROBIAL CONSORTIA USED FOR BIOREMEDIATION: BIOSYNTHESIS, FUNCTION AND REGENERATION Po-Hsiang Wang Doctor of Philosophy Department of Chemical Engineering and Applied Chemistry University of Toronto 2018 ABSTRACT Most microorganisms in nature live in communities and have developed tightly coordinated metabolism via metabolite exchanges. Cofactors are “helper molecules” in all cells, modulating the activity of many enzymes or serving as the electron shuttle. However, biosynthesis of cofactors, such as cobamides, is sometimes accomplished by only one specific phylogenetic group of microorganisms. Therefore, cofactors and their producers play pivotal roles in the functionality, metabolic rates, and population structure of a microbial community. This thesis focuses on three types of cofactors: (i) cobamides, (ii) NADPH, and (iii) prenylated flavin mononucleotide (prFMN) that are involved in anaerobic bioremediation of chlorinated solvents and aromatic pollutants. Cobamides are a family of cobalt-containing tetrapyrroles involved in biochemical reactions including methyltransfer reactions, isomerizations, and reductive dehalogenation. This thesis reports the identification of a functional cobamide prosthetic group in tetrachloroethene ii dehalogenases (PceA) of Desulfitobacterium hafniense and Geobacter lovleyi using a tiered blue-native polyacrylamide gel electrophoresis (BN-PAGE) and liquid chromatography-mass spectroscopy (LC-MS) method. The metabolic annotations of Dehalobacter restrictus (Dhb), a model organism for bioremediation of chlorinated solvents, were experimentally verified. The verified annotations were written into a constraint-based metabolic model, which identified that NADPH regeneration and de novo serine biosynthesis could be bottlenecks in Dhb metabolism. Using an integrated computational/experimental approach, the stringent nutrient requirements of Dhb were characterized. Further experimental analysis on the Dhb-enriched ACT-3 consortium has revealed an interspecies malate-pyruvate shuttle system between Dhb and its syntrophic partner. prFMN is a newly identified cofactor of UbiD reversible aromatic decarboxylases that are involved in ubiquinone biosynthesis, biological decomposition of lignin monomers, and anaerobic biodegradation of aromatic pollutants. We discovered that in Escherichia coli, dimethylallyl-monophosphate (DMAP), the prenyl donor of prFMN, can be produced from either prenol phosphorylation or from dimethylallyl-pyrophosphate dephosphorylation. In conclusion, this thesis reports biosynthesis and function of new cofactors as well as new mechanisms for reconciling cofactor regeneration in anaerobic microbial communities. Realizing these metabolic interdependencies generates opportunities to manipulate the microbiomes for better outcomes in bioremediation and industrial biotechnology. Interestingly, the identified interspecies cofactor exchange mechanism also provides insights into how life gradually evolved into complexity. iii ACKNOWLEDGEMENTS First of all, I would like to express my gratitude to all the people who have helped me in my life during these four and half years, especially my parents. Without their support, I could not make my dream, studying abroad, come true. Life is evanescent and is filled with surprises. I can still clearly remember the day I landed at Pearson International Airport. During my PhD study, I have met and collaborated with so many peers and professors in BioZone and at the University of Tennessee Knoxville, USA. Their help empowered me to finish my PhD and to write this thesis. Second, I want to acknowledge my supervisor, Prof. Elizabeth Edwards, for her unsparing contributions to my PhD study. While being strict, she never set up a hierarchy between us. She significantly improved my English speaking and writing skills, bridged my jumping ideas, and provided the research resources to satisfy my scientific curiosity. Moreover, she helped me realize the importance of communication and brought sophistication to my research. Furthermore, she always managed to expand my academic networks and paved the way for my future career. Third, I would like to acknowledge my committee members, Prof. Alexander Yakunin and Prof. Emma Master, and departmental examiner Prof. Radhakrishnan Mahadevan for their support and insightful suggestions on my thesis. I also acknowledge Prof. Frank Löffler, at University of Tennessee, USA, for teaching me the use of multiple lines of evidence approach to gain confidence in scientific hypothesis. Finally, friendship was an essential element in my PhD life. I would like to acknowledge all my friends, especially Dr. Shuiquan Tang, Dr. Fei Luo, Luz A. Puentes, Shen Guo, and Olivia Molenda in the Edwards Lab; Kayla Nemr, Naveen Venayak, and Kevin Correia in the Mahadevan Lab; Dr. Anna Khusnutdinova and Robert Flick in the Yakunin Lab; and Dr. Jun Yan in the Löffler Lab. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................................. ...IV TABLE OF CONTENTS…………………………………………………………………………………………………………….V LIST OF TABLES ............................................................................................................................. VII LIST OF FIGURES .......................................................................................................................... VIII LIST OF APPENDICES ........................................................................................................................ X LIST OF ABBREVIATIONS .............................................................................................................. XI CHAPTER 1 - LITERATURE REVIEW AND GENERAL INTRODUCTION ............................. 1 1.1 LITERATURE REVIEW ...................................................................................................................... 1 1.1.1 ORGANOHALIDE RESPIRATION ............................................................................................... 2 1.1.2 CORRINOIDS ......................................................................................................................... 11 1.1.3 NADPH: REGENERATION AND SHUTTLE SYSTEM ................................................................ 18 1.1.4 PRENYLATED FLAVINS AND REVERSIBLE DECARBOXYLATION OF AROMATICS ................... 21 1.2 RATIONALE AND RESEARCH OBJECTIVES..................................................................................... 23 1.3 THESIS OUTLINE AND STRUCTURE ............................................................................................... 25 1.4 STATEMENT OF AUTHORSHIP AND PUBLICATION STATUS ............................................................. 27 CHAPTER 2: IDENTIFICATION OF FUNCTIONAL COBAMIDE PROSTHETIC GROUP IN REDUCTIVE DEHALOGENASES USING BN-PAGE AND LC-MS ........................................... 31 2.1 ABSTRACT .................................................................................................................................... 31 2.2 INTRODUCTION ............................................................................................................................. 32 2.3 MATERIALS AND METHODS .......................................................................................................... 34 2.4 RESULTS ....................................................................................................................................... 37 2.5 CONCLUSION ................................................................................................................................ 41 CHAPTER 3: REFINED EXPERIMENTAL ANNOTATION REVEALS CONSERVED CORRINOID AUTOTROPHY IN CHLOROFORM-RESPIRING DEHALOBACTER ISOLATES ............................................................................................................................................ 43 3.1 ABSTRACT .................................................................................................................................... 43 3.2 INTRODUCTION ............................................................................................................................. 44 3.3 MATERIALS AND METHODS .......................................................................................................... 47 v 3.4 RESULTS AND DISCUSSION ........................................................................................................... 50 3.5 IMPLICATION FOR MICROBIAL ECOLOGY ..................................................................................... 68 CHAPTER 4: INTERSPECIES MALATE-PYRUVATE SHUTTLE DRIVES AMINO ACID EXCHANGE IN ORGANOHALIDE-RESPIRING MICROBIAL COMMUNITIES ................. 70 4.1 ABSTRACT .................................................................................................................................... 70 4.2 INTRODUCTION ............................................................................................................................. 71 4.3 MATERIALS AND METHODS .........................................................................................................
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