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Integration of Genome Content, Enzyme Activities, and Expression Profiles In Integration of Genome Content, Enzyme Activities, and Expression Profiles in Assessing Changes in End-Product Yields in Clostridium thermocellum by Thomas Rydzak A Thesis Submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Microbiology University of Manitoba Winnipeg Copyright © 2013 by Thomas Rydzak ii Integration of Genome Content, Enzyme Activities, and Expression Profiles in Assessing Changes in End-Product Yields in Clostridium thermocellum by Thomas Rydzak University of Manitoba, 2013 Supervisory Committee Dr. Richard Sparling (Department of Microbiology, University of Manitoba) Supervisor Dr. Ivan Oresnik (Department of Microbiology, University of Manitoba) Departmental Member Dr. Deborah Court (Department of Microbiology, University of Manitoba) Departmental Member Dr. Nazim Cicek (Department of Biosystems Engineering, University of Manitoba) Department Member Dr. David Wu (Department of Chemical Engineering, University of Rochester) External Examiner iii Supervisory Committee Dr. Richard Sparling (Department of Microbiology, University of Manitoba) Supervisor Dr. Ivan Oresnik (Department of Microbiology, University of Manitoba) Departmental Member Dr. Deborah Court (Department of Microbiology, University of Manitoba) Departmental Member Dr. Nazim Cicek (Department of Biosystems Engineering, University of Manitoba) Department Member Dr. David Wu (Department of Chemical Engineering, Universiy of Rochester) External Examiner iv Abstract Clostridium thermocellum is a fermentative, Gram-positive, thermophile capable of cellulosome-mediated breakdown of hemicellulose and simultaneous biofuels (ethanol and H2) production, and is thus an excellent candidate for consolidated bioprocessing. However, ethanol and/or H2 production yields are below theoretical maxima due to branched product pathways. Biofuel yields may be improved by manipulation of fermentation conditions or implementation of rational metabolic engineering strategies. However, the latter relies on a thorough understanding of gene content, gene product expression, enzyme activity, and intracellular metabolite levels, which can all influence carbon and electron flux. The thesis work represents the first large-scale attempt in combining bioinformatic, enzymatic, proteomic, and culture perturbation approaches to systematically understand these interactions. C. thermocellum was used to investigate how these parameters affect end-product yields. Enzyme activities involved in conversion of pyruvate to end-products were consistent with end-product profiles and draft genome annotation. NADH and NADPH- dependent alcohol dehydrogenase (ADH) activities were comparable, whereas NADPH- dependent hydrogenase activities were higher than NADH and ferredoxin-dependent hydrogenase activities. While product yields changed in response to exogenous end- product additions, most core fermentative enzyme activities did not, suggesting that these changes may be governed by thermodynamics. The lack of major changes (>2-fold) in expression in response to growth and gas sparging was further confirmed by proteomics and RT-qPCR, respectively, although the latter revealed that ADH expression changes in response to gas sparging. v Improved genome curation allowed refinement of metabolic pathways. A genomic and end-product meta-analysis of ethanol and/or H2 producing fermentative bacteria revealed that presence/absence of genes encoding hydrogenases and aldehyde dehydrogenases/ADHs had the greatest impacts on biofuel yields. However, genome content alone did not necessarily explain end-product yields. Given that genomic analysis of C. thermocellum revealed the presence of redundant genes encoding enzymes with analogous functions, shotgun and multiple reaction monitoring proteomics was used to refine which proteins are expressed. Absence/low expression of aldehyde dehydrogenase, ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase suggest that these enzymes may not play a significant role in metabolism. An alternative electron flow pathway is proposed to explain end- product synthesis patterns in response to pyruvate addition or presence of protein inhibitors (CO, hypophosphite). vi Acknowledgements The support, guidance, and friendships of so many people have made this endeavour possible. Firstly, I would like to thank my advisor, Dr. Richard Sparling, for his guidance, unwavering support, and the opportunity to persue this research. Thank you for challenging me, believing in me, and allowing me the flexibility and independence to branch out and peruse new challenges. I would also like to thank Dr. David B. Levin for his dedication and tireless efforts to ensure the success of this project. Your endless optimism has taught me to ‘persevere’ through challenging times. I would like to extend my appreciation to my supervisory committee members. Dr. Nazim Cicek, thank you for your worldly advice and showing me the importance of balancing life. Dr. Ivan Oresnik, thank you for always challenging me and teaching me to stand up for what I believe in. It has been both humbling and empowering. Finally, Dr. Deborah Court, thank you for your kindness and mentorship. Your classes were one of the reasons why I continued in this endeavour. Last but not least, my sincerest thanks to all of my collaborators, mentors, and lab members at the University of Manitoba. There have been too many to mention. Special thanks to John A. Wilkins and Oleg V. Krokhin for allowing me to pursue proteomics and work in your lab, Peyman Ezzati for your mentorship and technical support, Vic Spicer, for helping me sort through and make sense vast fields of data, Patric Chong, for teaching me the ropes, and Sharon Berg for all your help throughout the years. Thank you to all the members of our lab(s) for your friendship, help, support, and ideas: Carlo Carere, Tobin Verbeke, Marcel Taillefer, Scott Wushke, Umesh Ramachandran, Rumana Islam, Marina Grigoryan, Warren Blunt, Nathan Wrana, John Schellenberg, Peter McQueen, Zack Cunningham, Ian Cunningham, David Yu, and so many others that are too numerous to mention. Here’s to late nights and early mornings. vii Dedication To my parents, Grace and Henryk Thank you for teaching me the importance of hard work, challenging me, and having unconditional and unwavering faith in me, even when logic would disagree. And to my partner, Monika Thank you for your unbelievable patience and support. You have given me reason, stability, serenity, and sanity. You are my rock in an ocean full of woes. viii Table of Contents Abstract…… ..................................................................................................................... iv Acknowledgements .......................................................................................................... vi Dedication… .................................................................................................................... vii Table of Contents ........................................................................................................... viii List of Tables .................................................................................................................. xix List of Figures ................................................................................................................. xxi List of Supplementary Tables and Figures ................................................................ xxiii List of Abbreviations .................................................................................................... xxv Chapter 1: Literature Review ........................................................................................ 1 1.1 Introduction ............................................................................................................. 1 1.2 Rationale for biofuels .............................................................................................. 3 1.2.1 Economic and political pressures........................................................................ 3 1.2.2 Environmental concerns...................................................................................... 5 1.3 Bioethanol and biohydrogen ................................................................................... 7 1.3.1 Bioethanol ........................................................................................................... 8 1.3.2 Biohydrogen ...................................................................................................... 10 ix 1.3.2.1 Non-biological hydrogen production ........................................................ 11 1.3.2.2 Methods of biological hydrogen production ............................................. 14 1.4 Lignocellulose for biofuels production ................................................................ 16 1.4.1 First generation versus second generation biofuels .......................................... 16 1.4.2 Lignocellulose composition and pre-treatment ................................................. 17 1.4.3 Enzymatic cellulose and hemicellulose hydrolysis ........................................... 20 1.4.4 Consolidated bioprocessing for second-generation biofuels ............................ 25 1.5 Fermentative pathways of biofuel-producing cellulolytic bacteria................... 27 1.5.1 Oligosaccharide entry and breakdown .............................................................
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