UBC Theses and Dissertations
A systems biology approach to annotate novel open reading frames in the Saccharomyces cerevisiae Enoferm M2 wine yeast strain Ferguson, Andrew Taplin
Saccharomyces cerevisiae has been extensively studied both for its commercial applications in alcoholic beverages, bread and biofuel, and as a genetic model for higher eukaryotes; a function, however, has still not been described for over 10% of S. cerevisiae’s genes, and furthermore novel open reading frames (ORFs) have recently been identified in many yeast strains isolated from wine, beer and bioethanol industries. The present study used a systems biology approach, incorporating metabolomics, transcriptomics and proteomics, to systematically analyze novel ORFs and poorly-annotated genes during wine fermentation. Genes were either deleted or constitutively expressed in a commercial wine yeast, Enoferm M2, and the mutant strains were used to ferment Chardonnay grape must. Primary metabolites were monitored throughout the fermentation by high-performance liquid chromatography (HPLC), volatile metabolites were analyzed in the final wine by gas chromatography mass spectrometry (GCMS), and the transcriptome and proteome of the fermenting yeast were analyzed by ribonucleic acid (RNA) microarray and isobaric tagging for relative and absolute quantification (iTRAQ), respectively. The expression of fourteen novel ORFs from the Enoferm M2 genome was confirmed for the first time, and data was generated for the effect of mutation of these novel ORFs on wine fermentation. In addition to the novel ORFs, four poorly-annotated genes (YLL054C, YKL222C, PHD1 and HMS1) were found to positively regulate the allantoin metabolic pathway, and had additional effects on the cell wall and transmembrane transport. The GEP5 gene was found to negatively regulate the sulphur metabolic pathway, which could have significant effects on both sulphite tolerance and the production of off-flavours such as hydrogen sulphide during wine production. The systems biology approach used in conjunction with systematic gene mutation and industrially-relevant conditions was able to improve the annotation of novel and poorly-annotated genes in the S. cerevisiae genome.
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