Elucidating the metabolic pathways responsible for higher alcohol production in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancientmicrobiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae areusually responsible for most of the fermentative activity, and many data sets clearlydemonstrate the important impact of this species on the quality and character of the finalproduct. However, many aspects of the genetic and metabolic processes that take place duringalcoholic fermentation remain poorly understood, including the metabolic processes that impacton aroma and flavour of the fermentation product. To contribute to our understanding of theseprocesses, this study took two approaches:In a first part, the initial aim had been to compare two techniques of transcriptome analysis,DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability toassess wine fermentation gene expression changes, and in particular to assess their potentialto, in combination, provide combined quantitative and qualitative data for mRNA levels. TheSAGE methodology however failed to produce conclusive data, and only the results of themicroarray data are shown in this dissertation. These results provide a comprehensive overviewof the transcriptomic changes during model wine fermentation, and serve as a referencedatabase for the following experiments and for future studies using different fermentationconditions or genetically modified yeast.In a second part of the study, a screen to identify genes that impact on the formation of variousimportant volatile aroma compounds including esters, fatty acids and higher alcohols ispresented. Indeed, while the metabolic network that leads to the formation of these compoundsis reasonably well mapped, surprisingly little is known about specific enzymes involved inspecific reactions, the genetic regulation of the network and the physiological roles of individualpathways within the network. Various factors that directly or indirectly affect and regulate thenetwork have been proposed in the past, but little conclusive evidence has been provided. Togain a better understanding of the regulations and physiological role of this network, we took afunctional genomics approach by screening a subset of the EUROSCARF strain deletion library,and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higheralcohol formation were selected. Over-expression and single and multiple deletions of theselected genes were used to genetically assess their contribution to aroma production and tothe Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redoxhomeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight theimportant role of Bat2p in controlling the flux through the pathway.

[发布机构] Stellenbosch University