UBC Theses and Dissertations
Functional analysis of non-annotated genes in Saccharomyces cerevisiae using wine yeast and chardonnay fermentation Iwashita, Mayumi
The genome sequence of a laboratory Saccharomyces cerevisiae, S288c, was first released in 1996, and novel genes/ORFs continue to be discovered in other S. cerevisiae strains. Approximately one-third of genes/ORFs in S288c are still missing their biological process annotations. Gene functional studies using non-laboratory S. cerevisiae strains is a promising approach, as non-annotated genes/ORFs in laboratory S. cerevisiae are speculated to have lost phenotypes due to mutations during ~80 years of storage in laboratory conditions. The phenotypes of 149 non-annotated ORFs clustered as upregulated or expressed constitutively during wine fermentation (Marks et al. 2008) and eight novel ORFs that are conserved among wine strains but not found in S288c were investigated using a commercial wine S. cerevisiae called Enoferm M2 during Chardonnay fermentation. The overexpression of RGI1 and YDR249C resulted in increased acetic acid production (1.26-fold, p = 0.0491 and 1.52-fold increase, p = 0.001, respectively). Deletion of GEP5 resulted in higher glycerol (1.39-fold, p = 0.0005) and acetic acid (1.61-fold, p = 0.0020). The deletion of nine ORFs (GEP5, MTC7, PAR32, YBL071C-B, YCR051W, YDR089W, YDR249C, YDR524W-A, YMR027W) caused slow growth phenotypes in an aerobic growth curve analysis in Chardonnay grape must at three different temperatures (19, 22 and 30 °C). The localization of C-terminally GFP-tagged proteins was observed for GEP5, PDR18, YBL071C-B andYDR114C as well as five novel ORFs. One of the novel M2 ORFs, 13-2, is a potential novel transcription factor with a Zn(2)-C6 fungal-type DNA-binding domain and was found to localize in the nucleus. The deletion of 13-2 caused a reduction in the expression of ZRT1 by 5-fold (p = 0.0016) based on a qRT-PCR method. A search for additional novel ORFs in the Enoferm M2 genome sequence resulted in the identification of 16 novel ORFs, all of which were previously identified in non-reference S. cerevisiae genome sequences. The majority of non-annotated ORFs and novel ORFs investigated in this study did not show strong phenotypes during Chardonnay wine fermentation upon their deletions and/or overexpressions. It is speculated that conditions that are more challenging than the fermentation of table wine will be required to elucidate their functions.
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