Investigation and characterisation of highly nitrogen efficient wine yeast

Sufficient yeast assimilable nitrogen (YAN) is essential for wine yeast (Saccharomyces cerevisiae) to complete alcoholic fermentation. YAN largely consists of alpha-amino acids and ammonium ions. With adequate YAN (as well as other nutrients) in grape juice, yeast cellular processes such as protein synthesis, growth and proliferation occur, allowing alcoholic fermentation to proceed efficiently. By contrast, insufficient YAN may result in sluggish or stuck fermentation and is often coupled with the formation of undesirable aromas, such as hydrogen sulfide, which impact on wine quality. Employment of highly nitrogen efficient (HNE) wine yeast provides an alternative strategy to facilitate the completion of alcoholic fermentation under limited nitrogen conditions. In this study, a group of HNE candidate strains were investigated in both synthetic media and grape juice under different YAN conditions. A mutant with disruption of ECM33 showed superior fermentation performance under various YAN conditions compared with the wild type. Accordingly, the Δecm33 strain is defined as a HNE strain. The role of ECM33 was further investigated using the loss-of-function Δecm33 mutant. Growth on agar plates containing Calcofluor White (CFW) or Congo Red (CR) was limited, suggesting that Δecm33 possesses a cell wall defect resulting in increased chitin (target of the antifungals CFW and CR). QRT-PCR results showed that the transcriptional abundance of a group of key genes involved in the cell wall integrity (CWI), high osmolarity glycerol (HOG) and central nitrogen metabolism (CNM) pathways were altered in Δecm33 mutant. In order to understand the HNE mechanism in the Δecm33 strain, two genes PTP2 and SLT2 were investigated by overexpression, and also evaluated for their fermentation performance in synthetic media. Results showed that the overexpression of PTP2 improved yeast fermentation performance in the late stages of fermentation. SLT2 overexpression was not found to be helpful for fermentation. Metabolites examined in fermentation samples showed that a higher concentration of citric acid and ethanol were produced in PTP2 overexpression (OEX) strains. Increased ethanol yield was also observed in the SLT2 OEX strain. Less acetaldehyde was produced in the Δecm33 background strains during alcoholic fermentation. Based on the observations in this study, it is suggested that the HNE phenotype of Δecm33 might be triggered by activation of a metabolic network(s), including: one or more of the CWI, HOG or CNM pathways, resulting in a more robust yeast cell with good fermentative capability and adaption to the dynamic environment of alcoholic fermentation.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2014