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

217 Pages·2011·5.03 MB·English

by  Styger G

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

by
Gustav Styger

Dissertation presented for the degree of
Doctor of Philosophy (Science)

at
Stellenbosch University
Institute for Wine Biotechnology, Department of Viticulture and Oenology,
Faculty of AgriSciences

Promoter: Prof. F.F. Bauer

March 2011
Declaration

By submitting this dissertation electronically, I declare that the entirety of the work contained
therein is my own, original work, that I am the owner of the copyright thereof (unless to the
extent explicitly otherwise stated) and that I have not previously in its entirety or in part
submitted it for obtaining any qualification.

Date: 01/11/2010

Copyright © 2011 Stellenbosch University
All rights reserved
Summary

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.
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 oligo-
mikro-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.
This dissertation is dedicated to my parents
Biographical sketch

Gustav Styger was born in Roodepoort on the 3rd of October 1975. He attended Laerskool
Gustav Preller and matriculated in 1993 from Florida Hoërskool. Gustav obtained a B.Sc. cum
laude degree (Biochemistry, Zoology and Botany) from the Rand Afrikaans University (now
known as the University of Johannesburg) in 1996. In 1997 he obtained the degree B.Sc.
Honours cum laude (Biochemistry) from Stellenbosch University and in 2001 the degree M.Sc.
cum laude (Biochemistry) from the same institution.
Acknowledgements

I wish to express my sincere gratitude and appreciation to the following persons and institutions:
 Prof Florian Bauer – for endless encouragement, patience and positive thinking
 Prof Bernard Prior – for invaluable help with editing the thesis and contributing many
stimulating ideas
 Dr Dan Jacobson – for help with statistical analysis of not only the microarray dataset, but
the datasets from the other chapters as well
 Dr Heinrich Volschenk – for the opportunity to complete my experimental work in his
laboratory
 The Institute for Wine Biotechnology, including staff and students for giving me the
chance to complete my studies in an excellent academic environment
 All my lab colleagues and friends – for many happy memories both inside and outside
the lab
 National Research Foundation and Winetech - for funding
 My family – without whose support I could never have achieved this….
Preface

This dissertation is presented as a compilation of six chapters and an appendix. Each chapter
is introduced separately and is written according to a general style as Chapter 4 will be
submitted to the journal Applied Microbiology and Biotechnology and Chapter 5 will be
submitted for publication to the journal Applied and Environmental Microbiology

Chapter 1 General Introduction and Project Aims

Chapter 2 Literature Review
Wine Flavour and Aroma

Chapter 3 Research results
Transcriptome analysis of an industrial yeast strain during a model wine
fermentation: Results from oligo-cDNA microarrays

Chapter 4 Research results
Identifying genes that impact on aroma profiles produced by Saccharomyces
cerevisiae and the production of higher alcohols

Chapter 5 Research results
Genetic analysis of the metabolic pathways responsible for higher alcohol
production in Saccharomyces cerevisiae

Chapter 6 General discussion and conclusions

Appendix Lilly M., Bauer F.F., Styger G., Lambrechts M.G. & Pretorius I.S., (2006) The
effect of increased branched-chain amino acid transaminase activity in yeast on
the production of higher alcohols and on the flavour profiles of wine and
distillates. FEMS Yeast Research. 6(5) 726-743