Functional genomic analysis of sulfur and flavour-related yeast metabolism in wine fermentation with and without fining agents pectin and carrageenan

Abstract

Since the first vineyard was planted in Sydney more than 200 years ago, the Australian wine industry, propelled by technological advances in yeast biology, viticulture and the fermentation process, has been transformed into a multi-billion dollar economy for export and domestic consumption. Flavour, flavour stability and clarity are key characteristics of white wines. The wine flavour profile is primarily governed by secondary metabolites from fermenting yeast (Saccharomyces cerevisiae) and most of the flavoursome metabolites result from nitrogen metabolism, with many of the flavour compounds derived from amino acids. In another aspect, sulphur containing compounds, such as hydrogen sulfide (H2S) and sulfur dioxide (SO2) are metabolic by-products which can have a serious impact on fermented beverages. Clarity is one of the major constraints in the wine industry, since most of the methods available to avoid clarity problems such as hazing involve expensive or time consuming procedures that often lead to loss of positive flavour compounds. In this project, the molecular mechanisms underlying H2S and SO2 metabolism were investigated, and a new clarity-enhancing measure was explored and its effect on flavour profile analysed. The experimental approaches included anaerobic fermentation in both laboratory and pilot-scale fermenters, yeast cDNA gene expression microarray technology and solid phase microextraction gas chromatography - mass spectrometry (GC-MS SPME) for gene expression and volatile flavour profiling over time and between treatments. At the start of this PhD project, there were limited options in terms of yeast genome-wide differential gene expression microarray platforms. Due to the cost restraint and the need to outsource the one-colour Affymetrix chips, optimisation of glass-based two-colour cDNA microarray technology was carried out in-house. Many aspects of the protocol were examined such as cDNA method, dye type and hybridisation condition. As a result, however, only limited improvements were achieved. Since then, the outsourced more reliable one-colour Affymetrix chips became cheaper, and were therefore used as the microarray platform for subsequent experiments. The issue of replicate design was investigated in order to clarify whether biological triplicate microarray data were necessary if similar results were achievable in duplicates with lower costs. To verify this, microarray data that had been obtained in biological triplicate was analysed in a duplicate manner and the generated lists of significant genes were compared. Comparison of ANOVA analyses of the triplicate dataset and each of the three possible duplicate datasets showed R2 values of at least 0.95, indicating high correlation between these sets. Out of those genes that were significantly up- or down-regulated, three-quarters of the genes were common between the triplicate gene list and each of the duplicate sets, with the genes that weren't common being those with only minor differential gene expression. This suggests that yeast microarray experiments can be carried out in biological duplicates, saving a third of the costs without significantly changing the results. However, a major disadvantage is that p-value generation requires triplicate data. The analysis also demonstrated that technical replicates were unnecessary. H2S is an essential sulfur-containing compound found in wine. Growing conditions containing cysteine resulted in elevated H2S and SO2 production, however, when a nitrogen source was added, H2S was decreased. To elucidate the underlying molecular mechanism, microarray analysis was performed with a set of conditions including cysteine only, nitrogen (in the form of ammonium sulfate) only, combination of cysteine and nitrogen, and a control with neither cysteine nor nitrogen. The data analysis suggests that nitrogen catabolite repression (NCR) may be responsible for both the reduction in H2S quantities when the rich nitrogen source, ammonium, is present, but also could be linked with why yeast release H2S when grown in cysteinerich media. ABSTRACT

Date of Award	2012
Original language	English