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Meiotic Recombination-Based Genome Shuffling of Saccharomyces Cerevisiae and Characterization by Genome Sequencing and RNA-Seq T

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Meiotic Recombination-Based Genome Shuffling of Saccharomyces Cerevisiae and Characterization by Genome Sequencing and RNA-Seq T Meiotic recombination-based genome shuffling of Saccharomyces cerevisiae and characterization by genome sequencing and RNA-seq transcriptional expression profiling for improved tolerance to spent sulfite liquor ! ! Dominic Pinel A Thesis In The Department Of Biology Presented in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy at Concordia University ©Dominic Pinel, June 2013 ! CONCORDIA UNIVERSITY SCHOOL OF GRADUATE STUDIES This is to certify that the thesis prepared By: Dominic Pinel Entitled: Meiotic recombination-based genome shuffling of Saccharomyces cerevisiae and characterization by genome sequencing and RNA-seq transcriptional expression profiling for improved tolerance to spent sulfite liquor and submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Biology complies with the regulations of the University and meets the accepted standards with respect to originality and quality. Signed by the final examining committee: Dr. Joanne Turnbull Chair Dr. Shawn Mansfield External Examiner Dr. Paul Joyce External to Program Dr. Reginald Storms Examiner Dr. Malcolm Whiteway Examiner Dr. Vincent Martin Thesis Supervisor Approved by Dr. S. Dayanandan Chair of Department or Graduate Program Director June 6/2013 Dr. B. Lewis Dean of Faculty ! ABSTRACT Meiotic recombination-based genome shuffling of Saccharomyces cerevisiae and characterization by genome sequencing and RNA-seq transcriptional expression profiling for improved tolerance to spent sulfite liquor Dominic Pinel Ph. D. Concordia University, June, 2013. Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars that can be fermented to ethanol. However, the inhibitory substances found in this complex feedstock adversely affect yeasts used for the fermentation of the sugars in SSL. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome shuffling based on large-scale population recursive meiotic recombination. Populations of UV-induced yeast mutants more tolerant to hardwood spent sulfite liquor (HWSSL) were isolated and then recursively mated and enriched for more tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. These results demonstrate that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. The genome of R57, the most inhibitor-tolerant strain ! iii! ! generated in this study, was sequenced by massively parallel sequencing and twenty single nucleotide polymorphism mutations were located. Many of these mutations affect genes that correlate with known stress responses to the types of inhibition found in lignocellulosic hydrolysates. Cross-referencing the mutation findings with RNA-seq- derived differential gene expression analysis of R57 yields genes and biological processes that are likely playing determinant roles in the HWSSL tolerance trait of R57. The strongest findings support important roles for the following mutation-bearing proteins and biological processes: stress response transcriptional repressor, Nrg1p; NADPH-dependent glutamate dehydrogenase, Gdh1p, and a modified nitrogen usage and assimilation physiology including alterations to aromatic amino acid synthesis pathways and the associated Aro1p; protein homeostasis machinery including heat-shock 70-family proteins, especially Ssa1p, and Ubp7p and Art5p, which are related to ubiquitin-mediated proteolysis. Redox-associated metabolites NADPH, glutathione, mainly via GSH1 mutation, and iron are also implicated. Overall, these data provide important findings for understanding inhibition by multi-inhibitory lignocellulosic substrates; a meiotic recombination-mediated engineering strategy for generating inhibition-tolerant strains that may be unobtainable through classical evolutionary engineering; novel genetic targets for future rational biocatalyst design and inhibitor-tolerance studies; and a promising work-flow model for generating strains with desirable and complex phenotypic traits along with understanding of the genetic factors and biological processes involved in those traits. ! iv! ! Acknowledgement None of this work would have been possible without the support and comradery of the fine people in the Martin lab both past and present, many thanks. I particularly thank Euan Burton, Corinne Cluis, Nicholas Gold and Andrew Wieczorek who will always be family to me and who made Montreal a home. I acknowledge and thank my excellent committee members Reginald Storms and Paul Joyce, and Hung Lee, who was a brilliant collaborator and knowledgeable resource. Finally, I acknowledge my supervisor Vincent Martin who never let me down, opened all the right doors at all the right times, kept faith in me further than anyone could ask, and, most importantly, taught me how to be a scientist. ! v! ! Dedication This work is dedicated to my family. Your support, patience and love have made the difference. To my wonderful and supportive Gaby, who has stuck with me through this process despite the unfathomable miscalculations, your love means everything. To Juniper, who was the only one that could unwaveringly make me smile. To Eleanor, my hope for the future. To brother Chris, who was always there to listen to the frustrations and successes. To my father, Leo, who has made every step in my life possible and who unfailingly picked me up after every misstep. To my grandmother, Mary, who passed on during the writing of this thesis and who instilled in me a wonder of living things. And finally, to my mother, Bonnie, who was always my ardent supporter until the end, no matter where I chose to go and what I chose to do, she made me believe it was possible. ! vi! ! Table of contents List of tables ....................................................................................................................... xi List of figures .................................................................................................................... xii List of abbreviations ........................................................................................................ xiv Chapter 1 ............................................................................................................................. 1 1 Introduction ...................................................................................................................... 1 1.1 Project goals, objectives and hypotheses .................................................................. 1 1.1.1 Goal 1 ................................................................................................................. 1 1.1.2 Goal 2 ................................................................................................................. 2 1.1.3 Goal 3 ................................................................................................................. 2 1.2 Fermentation of lignocellulosic substrates to generate fuels or chemicals ............... 3 1.3 The effects of common sources of inhibition ............................................................ 5 1.4 Saccharomyces cerevisiae as a lignocellulosic bioethanol biocatalyst ..................... 6 1.5 Cellular response to fermentation inhibition ............................................................. 7 1.5.1 Global stress responses ....................................................................................... 7 1.5.2 Cellular response to specific lignocellulose hydrolysate inhibitors ................. 12 1.5.2.1 Furan aldehydes ......................................................................................... 12 1.5.2.2 Acetic acid ................................................................................................. 14 1.5.2.3 Phenolics .................................................................................................... 15 1.5.2.4 Osmotic stress ............................................................................................ 16 1.5.2.5 Ethanol tolerance and fermentation adaptation .......................................... 17 1.6 Spent sulfite liquor as an existing industrial inhibitory lignocellulosic substrate ... 19 1.7 Genome shuffling as a means to generate microbial strains displaying complex traits ............................................................................................................................... 21 1.8 Massively parallel DNA sequencing as a means to track random genomic changes ....................................................................................................................................... 24 1.8.1 MPS technology and detecting genome variations ........................................... 25 1.8.2 Bioinformatics resources for analyzing MPS genome sequence reads ............ 29 1.9 RNA-seq transcriptomics as an MPS-based tool for gene expression analysis ...... 30 1.10 Conclusions ........................................................................................................... 34 Chapter 2 ..........................................................................................................................
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