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UC Santa Barbara Dissertation Template UNIVERSITY OF CALIFORNIA Santa Barbara Developing a systems biology framework to engineer anaerobic gut fungi A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemical Engineering by St. Elmo Wilken Committee in charge: Professor Michelle A. O’Malley, Co-chair Professor Linda R. Petzold, Co-chair Professor M. Scott Shell Professor Elizabeth G. Wilbanks September 2020 The dissertation of St. Elmo Wilken is approved. ____________________________________________ Elizabeth G. Wilbanks ____________________________________________ M. Scott Shell ____________________________________________ Linda R. Petzold, Committee co-chair ____________________________________________ Michelle A. O’Malley, Committee co-chair September 2020 Developing a systems biology framework to engineer anaerobic gut fungi Copyright © 2020 by St. Emo Wilken iii ACKNOWLEDGEMENTS First, I would like to acknowledge and thank God for giving me the opportunity and ability to pursue my Ph. D. at UCSB. Next, I would like to thank my advisors, Michelle O’Malley and Linda Petzold for their guidance, support and encouragement in completing the work that went into this thesis. They always supported me in my desire to explore new avenues of inquiry and were patient when things did not work out as expected. The Dow Discovery Fellowship was also instrumental in the research freedom I had, and I gratefully acknowledge its support. I would also like to acknowledge the entire O’Malley lab, but specifically Susanna Seppälä and Tom Lankiewicz for their many insightful discussions about biology and their patience in explaining it to an engineer. I would also like to thank Jon Monk for guiding me through the process of developing the first genome-scale model of a decidedly non-model anaerobic gut fungus. I would not have been able to complete my Ph. D. without the support of my family, and specifically my parents and brother. They were always there for me, ready to listen and provided much needed emotional support as I completed my degree so far away from home. I would also like to acknowledge my friends, and specifically George Degen, for all the fun times we had in California. Most of all I want to thank my wife, Sina, for all her support and encouragement. She endured three years of a long-distance relationship, as well as all the highs and lows associated with completing a Ph. D., all the while being a constant source of strength. Thank you for everything you did for me! iv VITA OF St. Elmo Wilken September 2020 EDUCATION 2015 – 2020 Doctor of Philosophy in Chemical Engineering University of California, Santa Barbara 2017 – 2020 Graduate Program in Management Practice Certificate University of California, Santa Barbara 2014 – 2015 Master of Chemical Engineering, Control Engineering The University of Pretoria, Pretoria With distinction 2013 – 2014 Bachelor of Science Honors, Applied Mathematics The University of Pretoria, Pretoria With distinction 2012 – 2013 Bachelor of Engineering Honors, Control Engineering The University of Pretoria, Pretoria With distinction 2009 – 2012 Bachelor of Engineering, Chemical Engineering The University of Pretoria, Pretoria With distinction PROFESSIONAL EMPLOYMENT 2015 – 2020 Graduate Research Fellow and Teaching Assistant Department of Chemical Engineering, UC Santa Barbara Research Advisors: Michelle A. O’Malley & Linda R. Petzold 2013 – 2015 Graduate Researcher Department of Chemical Engineering Research Advisors: Carl Sandrock & Pieter J. de Villiers 2011 Chemical Engineering Intern Sublime Technologies, South Africa 2010 Chemical Engineering Intern Nuclear Energy Company of South Africa, South Africa v PUBLICATIONS 1. St. Elmo Wilken, Patrick Leggieri, Corey Kerdman-Andrade, Matthew Reilly, Michael K. Theodorou, Michelle A. O’Malley, An Arduino based Automatic Pressure Evaluation System (A-APES) to quantify growth of non-model anaerobes in culture, AIChe Journal (2020) 2. St. Elmo Wilken, Susanna Seppälä, Thomas S. Lankiewicz, Mohan Saxena, John K. Henske, Asaf A. Salamov, Igor V. Grigoriev, Michelle A. O’Malley, Genomic and proteomic biases inform metabolic engineering strategies for anaerobic fungi, Metabolic Engineering Communications (2019) 3. Sean P. Gilmore, Thomas S. Lankiewicz, St. Elmo Wilken, Jennifer L. Brown, Jessica A. Sexton, John K. Henske, Michael K. Theodorou, David L. Valentine, Michelle A. O'Malley, Top-down enrichment guides in formation of synthetic microbial consortia for biomass degradation, ACS Synthetic Biology (2019) 4. St. Elmo Wilken, Candice L. Swift, Igor A. Podolsky, Susanna Seppälä, Michelle A. O'Malley, Linking “omics” to function unlocks the biotech potential of non-model fungi, Current Opinion in Systems Biology (2019) 5. St. Elmo Wilken, Mohan Saxena, Linda R. Petzold, Michelle A. O'Malley, In Silico Identification of Microbial Partners to Form Consortia with Anaerobic Fungi, Processes (2018) 6. John K. Henske, St. Elmo Wilken, Kevin V. Solomon, Chuck R. Smallwood, Vaithiyalingam Shutthanandan, James E. Evans, Michael K. Theodorou, Michelle A. O'Malley, Metabolic characterization of anaerobic fungi provides a path forward for two-stage bioprocessing of crude lignocellulose, Biotechnology and Bioengineering (2017) 7. Susanna Seppälä*, St. Elmo Wilken*, Doriv Knop, Kevin V. Solomon, Michelle A. O'Malley, The importance of sourcing enzymes from non-conventional fungi for metabolic engineering & biomass breakdown, Metabolic Engineering (2017) *equal author contributions PUBLICATIONS IN PREPARATION OR IN REVISION 1. Xuefeng Peng, St. Elmo Wilken, Thomas S. Lankiewicz, Sean P. Gilmore, Jennifer L. Brown, John K. Henske, Candice L. Swift, Asaf Salamov, Kerrie Barry, Igor V. Grigoriev, Michael K. Theodorou, David L. Valentine, Michelle A. O’Malley, Sculpting gut microbial communities alters fermentation products and methane release, In revision at Nature Microbiology 2. St. Elmo Wilken, Jonathan M. Monk, Patrick A. Leggieri, Christopher Lawson, Thomas S. Lankiewicz, Susanna Seppälä, Stephen J. Mondo, Kerrie W. Barry, Igor V. Grigoriev, John K. Henske, Michael K. Theodorou, Bernhard O. Palsson, Linda R. Petzold, Michelle A. O’Malley, Experimentally validated reconstruction and analysis of a genome-scale metabolic model of an anaerobic Neocallimastigomycota fungus, In preparation for mSystems Journal vi SELECTED ORAL AND POSTER PRESENTATIONS 1. St. Elmo Wilken, Linda R. Petzold, Michelle A. O’Malley (2019). Developing a genome-scale model of Neocallimastigomycota fungi as a platform for metabolic engineering. Graduate Student Symposium at the University of California Santa Barbara, October 4th. Santa Barbara, California, USA. (Best speaker award) 2. St. Elmo Wilken, Linda R. Petzold, Michelle A. O’Malley (2018). Incorporating Flux Sampling into a Minimal Assumption Dynamic Flux Balance Analysis Algorithm. 5th Constraint-Based Reconstruction and Analysis Conference, October 14th – 16th. Seattle, Washington, USA. 3. St. Elmo Wilken, John K. Henske, Francis Cunningham, Michelle A. O’Malley (2017). Coarse Grained Model Development of Fungal Enzymatic Lignocellulosic Biomass Degradation. American Chemical Society Biotechnology Division, April 2nd – 6th. San Francisco, California, USA. SELECTED HONORS AND AWARDS 2017-2020 Dow Discovery fellowship award 2019 Best speaker award at UCSB’s chemical engineering graduate student symposium 2017 Distinguished service award from the University of California Santa Barbara’s chemical engineering department 2014 South African Mathematical Society’s award for the best honors student at the University of Pretoria 2014 Allan Gray Academic Achiever award 2012 Best undergraduate research project award from the University of Pretoria’s chemical engineering department vii ABSTRACT Developing a systems biology framework to engineer anaerobic gut fungi by St. Elmo Wilken Lignocellulose is a complex, energy-rich heterogenous polymer composed of cellulose (40-50%), hemicellulose (20-40%) and lignin (20-35%). Using the more than 1.6 billion tons of agricultural lignocellulosic waste generated worldwide each year is a promising avenue to explore for sustainable bioprocessing. While lignocellulose is abundant, lignin acts as a protective barrier that prevents its decomposition into fermentable sugars and poses significant challenges for the utilization of this energy-rich resource in biotechnological applications. On the other hand, anaerobic gut fungi specialize in bio-converting unpretreated lignocellulose into fermentable sugar monomers and represent a promising opportunity to exploit lignocellulosic plant biomass for bioprocesses. Anaerobic gut fungi typically inhabit the digestive tracts of herbivores where they play an integral role in the decomposition of raw lignocellulose into its constitutive sugar monomers. The genomes of these fungi encode for the highest diversity, and largest number, of lignocellulolytic enzymes of any sequenced fungus to date. This, in combination with their filamentous morphology, causes them to excel at lignocellulose decomposition. Despite these advantages, anaerobic gut fungi are not utilized in bioprocesses due to challenges in viii cultivating and engineering them. In this thesis a marriage between experimental and multi- omic datasets is used to develop techniques that can be used to better understand and engineer anaerobic gut fungi for lignocellulose decomposition in upstream bioprocesses. A comprehensive metagenomic enrichment of goat fecal pellets was undertaken to better understand how
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