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

Styger, Gustav (2011-03)

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011.

Includes bibliography.

Thesis

ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, 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 to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional 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 higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway.

AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.

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