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Clostridium Thermocellum Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2011 Model-Guided Systems Metabolic Engineering of Clostridium thermocellum Christopher Gowen Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Engineering Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/2529 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. ©Christopher M Gowen 2011 All rights reserved. MODEL-GUIDED SYSTEMS METABOLIC ENGINEERING OF CLOSTRIDIUM THERMOCELLUM A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT VIRGINIA COMMONWEALTH UNIVERSITY. BY CHRISTOPHER MARK GOWEN M.S. ENGINEERING, VIRGINIA COMMONWEALTH UNIVERSITY, 2008 B.S. BIOSYSTEMS ENGINEERING, CLEMSON UNIVERSITY, 2006 DIRECTOR: STEPHEN S. FONG, PH.D. ASSISTANT PROFESSOR, CHEMICAL & LIFE SCIENCE ENGINEERING VIRGINIA COMMONWEALTH UNIVERSITY RICHMOND, VIRGINIA MAY, 2011 ii ACKNOWLEDGEMENT For Grandy. "Can't never did do nothin'" I have many people to whom I am indebted for incredible love, support, and guidance. To begin, I would like to thank my advisor, Dr. Fong, for his guidance, instruction, and flexibility. Few educators can balance so well the sometimes competing drives for impactful research and scientific education, and I am grateful for the patience and flexibility he shows in letting his graduate students find their own independence. I would also like to thank Dr. Sherry Baldwin, Dr. Paul Brooks, Dr. Mark McHugh, Dr. Raj Rao, and Dr. Vamsi Yadavalli, who have also served quite a lot of time advising, challenging, and instructing me as members of my committee. I have also had the pleasure of working with too many fantastic friends and colleagues over the past five years to list here, and I am indebted to all of them for good advice, commiseration, and fun times. Thanks especially to Jack for his patience and good humor when navigating jungles of paperwork, to Cindy our lab Mom for keeping us all in line and being a good friend, and to my friends and labmates (in order of appearance!), Advait, Oscar, Deng, George, and Niti for all of the fun times that will not be spoken more of here. Finally, I am hugely blessed to have a loving and supportive family who has always encouraged me to ask questions, and to “just use your brain for a second and figure it out.” You all know who you are. At last, thanks to my beautiful wife Cathy who has patiently (!) endured uncertainty, boring science talk, odd hours, and yet more uncertainty. Thank you for all the times you have picked up my slack. iii CONTENTS A. General abbreviations ......................................................................................................... xii B. Reactions referenced in the text (a complete list is available in the appendix): .............. xiii I. Introduction ............................................................................................................................. 1 A. Systems biology: the informatics challenge ........................................................................ 1 B. In silico metabolic reconstructions ...................................................................................... 2 C. Cellulosic ethanol production .............................................................................................. 3 1. Motivation ........................................................................................................................ 3 2. Production strategies ....................................................................................................... 4 3. Clostridium thermocellum metabolic engineering .......................................................... 5 D. Dissertation overview .......................................................................................................... 5 II. Construction of a Genome-Scale Model of Clostridium thermocellum .................................. 8 A. Introduction ......................................................................................................................... 8 B. Methods ............................................................................................................................... 9 1. Construction of an in silico genome-scale stoichiometric model of C. thermocellum metabolism .............................................................................................................................. 9 2. Cell growth ..................................................................................................................... 14 3. Assay for succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) enzymatic activity ................................................................................................................................... 15 4. Model analysis ................................................................................................................ 15 C. Results ................................................................................................................................ 21 1. Model Description .......................................................................................................... 21 iv 2. Comparison of model results to experimental results .................................................. 28 3. Gene Deletions: essentiality and effects on ethanol secretion ..................................... 32 4. Tradeoff of H2 and ethanol production .......................................................................... 34 5. Comparative Analysis of metabolisms – compare to C. acetobutylicum and S. cerevisiae ............................................................................................................................... 36 6. Simulation of Alternative Media Formulations .............................................................. 37 D. Modifications to the original iSR432 model ...................................................................... 39 1. Pyruvate kinase .............................................................................................................. 40 2. Bifurcating hydrogenase and electron transport ........................................................... 41 E. Discussion........................................................................................................................... 43 III. Whole Transcriptome Sequencing of C. thermocellum on Cellobiose .............................. 44 A. Introduction ....................................................................................................................... 44 B. Background ........................................................................................................................ 45 1. Types of mRNA-level “omics” datasets .......................................................................... 45 2. Whole-transcriptome sequencing analysis (RNAseq) .................................................... 46 C. Methods ............................................................................................................................. 47 1. Sample Preparation ........................................................................................................ 47 2. Analysis of raw data ....................................................................................................... 48 D. Results ................................................................................................................................ 49 1. Distribution of gene expression levels ........................................................................... 49 2. Operon structure ............................................................................................................ 52 3. Transcriptional start sites ............................................................................................... 55 v E. Discussion........................................................................................................................... 59 IV. Computational Integration of Transcriptome and Proteome with the Genome-Scale Model of C. thermocellum ............................................................................................................ 61 A. Introduction ....................................................................................................................... 61 B. Methods ............................................................................................................................. 62 1. Boolean restriction of solution space ............................................................................ 62 2. Ruppin algorithm ............................................................................................................ 62 3. Evaluation of flux prediction quality .............................................................................. 63 C. Results ................................................................................................................................ 64 1. Algorithmic integration of RNAseq with metabolic model ............................................ 64 2. Comparison to other data integration methodologies .................................................
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