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Identifying Genes That Confer Ethanol Tolerance in Saccharomyces Cerevisiae

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Identifying Genes That Confer Ethanol Tolerance in Saccharomyces Cerevisiae Identifying genes that confer ethanol tolerance in Saccharomyces cerevisiae A thesis submitted for the degree of Doctor of Philosophy Tina Thi My Tien Tran Bachelor of Applied Biology (Biotechnology/Microbiology) - Honours 2011 The Australian Wine Research Institute School of Molecular Sciences Faculty of Health, Engineering and Science Wine Innovation Cluster Hoppers Lane, Werribee Corner of Hartley Grv and Paratoo Rd Melbourne Urrbrae Victoria South Australia DECLARATION “I, Tina Thi My Tien Tran, declare that the PhD thesis entitled ‘Identifying genes that confer ethanol tolerance in Saccharomyces cerevisiae’ is no more than 100,000 words in length including quotes and exclusive of tables, figures, appendices, bibliography, references and footnotes. This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma. Except where otherwise indicated, this thesis is my own work”. Signature Date i ACKNOWLEDGMENTS I am most grateful to my mentor and supervisor, Dr. Paul Chambers. Thank-you for being such a patient, understanding, and compassionate person. You generously donated a great deal of time and energy to mentor me. I would be lost without your valuable guidance and direction throughout my entire candidature. I am very grateful for your in-depth knowledge and willingness to share it with me. You have gone above and beyond to help me in my personal and professional development, and I am extremely thankful for your kindness. I would also like to thank my supervisor Professor Grant Stanley who has been my mentor since my undergraduate years. Your encouragement and support throughout my PhD candidature will always be remembered. I have been very blessed to have you both as my lecturers, supervisors, and mentors. Thank-you to my supportive friends in Melbourne and Victoria University, especially Dr. Dragana Stanley for having faith in me to continue your project, I appreciate your openness in sharing your data and friendship. To Dr. Anthony Borneman, thank-you for all your guidance and invaluable knowledge. I have learnt a great deal from you and would not have been able to complete this thesis without your help. To Dr Simon Schmidt, thank-you for your valuable input throughout the years, Your constant willingness to lend a hand or ear, is most appreciated. Thank-you for donating your valuable time to read my thesis and give me valuable feedback. To Mr. Angus Forgan, thank-you for being so understanding and supportive. I have always enjoyed having a chuckle and learning new things from you. Xin ciào Mrs. Jenny Bellon, Cám ơn for teaching me GLP. To Aunty June Robinson, thank-you for your friendship, encouragement and support. To the AWRI staff, in particular Prof. Sakkie Pretorius, Dr. Markus Herderich, Mr. Hans Muhlack, and Ms. Linda Halse, thank-you for your immense support, understanding and giving me the privileged opportunity to work at the AWRI during my PhD. ii To my fellow AWRI Wine Biosciences Team, particularly Dr. Eveline Bartowsky, Dr. Paul Henschke, Dr. Toni Cordente, Dr. Darek Kutyna, Ms. Jane McCarthy, Dr. Peter Costello, Dr. Cristian Varela, Ms. Robyn Kievit, Ms. Caroline Abrahamse, Ms. Gal Winter and Ms. Corine Ting, Thank-you for your friendship and much needed coffee breaks over these past few years. You have all made working at the AWRI, one of the best experiences of my life. Thank-you to the Grape and Wine Research Development Corporation (GWRDC) and Victoria University, for their generous financial support during my PhD candidature. To Mary and Gerry Rigter, you have been my family away from home. The generosity, kindness and love you have shown The Tran Family, will be in our hearts always. To My Family; my father, Nam Van Tran and my mother Lang Thi Nguyen, I cannot articulate the extent of how grateful I am to have you both. You have always had the utmost faith in me; your encouragement and unconditional love, gave me strength and hope. You have both sacrificed so much for us and I am forever indebted to you both. To my brothers Sang Tran, Dung Tran and Peter Tran, thank-you for making me laugh when I needed to most and for going above and beyond to take care of me. You guys have been there for me through all my trials and tribulations. To my extended family in Phu Quoc Island, especially Bà ngoại, your courage is inspirational. I am blessed to have you all in my life, sincere thanks for all the generosity, understanding, support, kindness and love that you have shown me over the years. I would not have been able to complete this thesis without you all. This has been a very rewarding experience for me both personally and professionally. Thank you all from the bottom of my heart. iii ABSTRACT S. cerevisiae has evolved the ability to tolerate high concentrations of ethanol, a trait that has contributed to this yeast being a cornerstone of beverage and biofuel industries. The key genes involved in conferring ethanol tolerance in S. cerevisiae are unknown. One strategy used to identify genes conferring ethanol tolerance, was to create ethanol-tolerant (ET) mutants from a laboratory yeast strain and use these to identify ethanol-tolerance conferring loci. Dr. Dragana Stanley (Victoria University) created two ET mutants, a spontaneous (SM) and a chemical (CM) mutant from W303, via adaptive evolution (Stanley 2008). Transcriptome analysis of the resultant ET mutants found that expression levels of hundreds of genes were altered, relative to the parent under ethanol-stress conditions. The primary objective of this thesis was to identify genes that confer ethanol tolerance in SM and CM. As a starting point, the mutants backcrossed and aporulated to characterize the genetics of the ethanol-tolerance mutations. A Rapid Ethanol Tolerance Assay (RETA) was developed from this work, enabling accurate quantification ethanol tolerance levels in the numerous progeny and spores generated. The segregation ratios of ethanol tolerance in CM indicated that a single gene was responsible for conferring the phenotype. In SM, ethanol tolerance segregated in a less straightforward fashion, suggesting more than two genes were responsible for conferring the phenotype. In attempt to identify the genetic mutations conferring ethanol tolerance in the mutants, Affymetrix Tiling Microarrays were applied. However, this method generated large amounts of background noise and was unable to resolve the mutations. Genomis sequencing of CM was then used revealing a large number of mutations across the genome. Candidate loci were screened, leading to identification of an intergenic region containing four SNPs, and this was found to confer ethanol tolerance when transformed into the parent. This Ethanol Tolerance Conferring Sequence (ETCS), was further characterized to reveal that only SNP1 (Chromosome II: 697,850 – SNP1 = C→A) and SNP3 (Chromosome II: 697,907 – SNP1 = C→T) were required to confer ethanol tolerance. iv Interestingly, ETCS resides in a transcribed, intergenic region between ORFs YBR238C and ERT1, genes which have not been previously associated with ethanol tolerance. Upstream of ERT1 is THI2, and transcriptome data from Stanley et al (2010) indicated that expression of these two genes is up-regulated under ethanol-stress conditions in CM. The intergenic region is highly conserved across a number of industrial S. cerevisiae strains. When industrial wine strains were made homozygous for ETCS, it was found that the effect of ETCS on the ethanol tolerance phenotype was genetic background dependent. Future work is required to elucidate the mechanism by which ETCS confers the ethanol tolerance in S. cerevisiae. v PUBLICATIONS AND PRESENTATIONS Publication Liccioli, T*., Tran T. M. T.,*., Cozzolino D., Jiranek V., Chambers P. J., & Schmidt S. A., “Microvinification – how small can we go?”, Applied and Environmental Microbiology, 89 (5):1621-1628 (2010) * Liccioli, T. and Tran T. M. contributed equally to the article. Conference Presentation Tran T. M. T ., Stanley D., Schmidt S. A., Bellon J. R., Borneman A., Stanley G. A., and Chambers P. J., “Identifying and Characterizing genes that confer the ethanol tolerance phenotype in Saccharomyces cerevisiae”, Yeast Products and Discovery, Adelaide, Australia (2009) Conference Poster Tran T. M. T., Stanley D., Schmidt S. A., Bellon J. R., Borneman A., Stanley G. A., and Chambers P. J., “Identification of genes that confer ethanol tolerance in Saccharomyces cerevisiae”..XXIIIrd International Conference on Yeast Genetics and Molecular Biology, Melbourne, Australia, 2007 Note An electronic PDF version of the thesis is located in the Appendix CD. vi ABBREVIATIONS Abbreviation Shortened Word % v/v Percentage volume per volume bp Base pair CIP Calf Intestinal Phosphatase CORE COunterselectable REporter DIG Digoxygenin EDTA Ethylenediaminetetraacetic acid EMS Ethylmethane Sulphonate ET Ethanol Tolerant ETCS Ethanol Tolerance Conferring Sequence gDNA Genomic Deoxyribonucleic Acid hrs Hours ITS Inter-transcribed Spacer LB Luria Broth min Minutes nET not Ethanol Tolerant PCI Phenol Chloroform Iso-amyl alcohol PEG Polyethylene Glycol PMPP Plasma Membrane Proton Pump RETA Rapid Ethanol Tolerance Assay SC Synthetic Complete SGD Saccharomyces Genome Database SNPs Single Nucleotide Polymorphisms SSC Saline Sodium Citrate ssDNA Salmon Sperm DNA TBE Tris Borate EDTA TE Tris-EDTA VIS Visible YPD Yeast Peptone Dextrose YPG Yeast Peptone Glycerol vii TABLE OF CONTENTS IDENTIFYING GENES THAT CONFER ETHANOL TOLERANCE IN SACCHAROMYCES
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