Analysis of Saccharomyces cerevisiae deletion mutants displaying a modified carbon flux under wine fermentative conditions

Madlanga, Ncedile Hamilton (Stellenbosch : University of Stellenbosch, 2009-03)

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009.

Thesis

Saccharomyces cerevisiae has been used for millennia for the leavening of dough and in the production of alcoholic beverages such as beer and wine. More recently, it is being used as cell factories for the production of important pharmaceutical products. S. cerevisiae has also been extensively used as a model organism for studying many genetic and biochemical processes within the eukaryotic cell. Since the completion of a yeast genome sequence, many functional analysis projects have emerged with the aim of elucidating the functions of the unidentified genes revealed by the genome sequence. One of the most relevant approaches consisted in the construction of a collection of mutants deficient in all single genes, either in a haploid background for non-essential genes, or as heterozygous diploids for essential genes. This collection of strains can be subjected to phenotypic screens that might reveal the function of unknown genes or add to our understanding of already annotated genes. While this approach is promising, it also bears some limitations. For instance, many mutants have no overt phenotypes and some phenotypes do not obviously showcase the function of the encoded protein. In this study, S. cerevisiae strains with single deletions of genes involved in pyruvate metabolism were selected from the Euroscarf deletion library. Pyruvate is a central intermediate of glycolysis, and pyruvate metabolism largely defines the general distribution of carbon flux in the cell. These mutants were screened for modified fermentation kinetics or modified carbon flux under wine fermentative conditions, an environment that had not been previously used for the analysis of these mutants. A strain disrupted in the PDA1 gene, which encodes the E1α subunit of the pyruvate dehydrogenase showed a significant change in phenotype when grown in wine fermentative conditions. In particular, the mutant displayed a prolonged lag phase, but upon entering exponential growth, fermented significantly faster than the wild type strain and completed alcoholic fermentation in a shorter period of time. This phenotype could be of significant industrial interest. The mutant phenotype was further investigated through disruption of the gene in the same as well as in different genetic backgrounds, and through complementation of the PDA1 deletion with a plasmid-born wild type copy. The data show that the PDA1 gene disruption is not solely responsible for the observed phenotypes under wine fermentative conditions. We therefore propose that secondary mutations have contributed to the mutant phenotype. This study shows that phenotypes attributed to a specific gene in mutants of the Euroscarf library should always be confirmed before performing consequent experiments and drawing significant conclusions.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/2158
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