Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The production of glycerol by Saccharomyces cerevisiae under anaerobic conditions is
essential for maintaining the intracellular redox balance thereby allowing continuous
energy generation through conversion of sugars into ethanol. In addition, glycerol can
act as an osmolyte and is synthesized to maintain turgor pressure under hyperosmotic
conditions. The production of ethanol from sugars can be a redox-neutral process,
where the NAD+ (nicotinamide adenine dinucleotide) that is consumed during the
glycolytic conversion of glyceraldehyde-3-phosphate to pyruvate is later regenerated by
the reduction of acetaldehyde to ethanol. However, in particular the redirection of
metabolic flux of pyruvate to biomass formation leads to excess NADH formation. The
intracellular redox balance in these conditions is then primarily maintained through
formation of glycerol which is control by two main enzymes, namely Gpd1p and Gpd2p.
Deletion of the genes coding for these two proteins leads to accumulation of NADH and
renders the cells incapable of maintaining their fermentative ability and growth under
anaerobic conditions.
The goal of this study was to investigate the growth, fermentative ability and metabolite
synthesis of various gpd1Δgpd2Δ double mutant (DM) strains in which the redox
balancing potential was partially restored through expression of native or heterologous
genes. Strains were constructed by introducing alternative NADH oxidizing pathways or
manipulating existing pathways to favour the oxidation of excess NADH. More
specifically, the modifications included (i) sorbitol formation; (ii) establishing a pathway
for propane-1,2-diol formation; and (iii) increasing ethanol formation. Apart from
genetically manipulating the gpd1Δgpd2Δ double mutant, the addition of pyruvate
during growth was also investigated. The experiments were carried out under oxygen
limited conditions in a high sugar medium and the fermented product was analyzed for
total sugar consumed, biomass and primary and secondary metabolites formed by the
different strains. The relationships between sugar consumption, growth and metabolite
production by different strains were investigated by comparing the data generated from
the different strains by using multivariate data analysis tools. Analysis of the pathways
involved in the production of primary (acids, ethanol and other metabolites) and
secondary metabolites (aroma compounds) were also carried out in order to establish
flux modification in comparison to the wild type (WT) strain. The results revealed that these manipulations improved the fermentative capacity of the
gpd1Δgpd2Δ double mutant, suggesting a partial recovery of NAD+ regeneration ability,
albeit not to the extent of the WT strain. As expected a significant correlation was found
between sugar consumption and ethanol and biomass formation. Ethanol yields but not
final concentrations were increased by the genetic manipulations. Sorbitol by DM(srlD)
and DM(SOR1) strains and propane-1,2-diol by DM(gldA, GRE3, mgsA) strain were
formed in significant amounts although at lower molar yields than glycerol.
Furthermore, by genetic manipulation the yield of secondary metabolites (isobutanol,
isoamyl alcohol, 2-phenyl ethanol and isobutyric acid) was increased whereas the ethyl
acetate concentration and yield decreased. The results indicate that aroma compound
properties of wine yeasts could be favourably changed by manipulating the glycerol
synthesizing pathway. The addition of pyruvate during the growth of gpd1Δgpd2Δ
double mutant contributes to excess NADH re-oxidation through additional ethanol
formation. / AFRIKAANSE OPSOMMING: Die produksie van gliserol deur Saccharomyces cerevisiae onder anaërobiese
toestande is noodsaaklik vir die onderhouding van die intrasellulêre redoksbalans en
maak dus ononderbroke energie-ontwikkeling tydens die omsetting van suikers in
etanol moontlik. Daarbenewens kan gliserol as ‘n osmoliet optree en word dit
gesintetiseer om turgordruk onder hiperosmotiese toestande te onderhou. Die
produksie van etanol uit suikers kan ‘n redoksneutrale proses wees, waar die NAD+
(nikotinamiedadenien-dinukleotied) wat tydens die glikolitiese omskakeling van
gliseraldehied-3-fosfaat na piruvaat verbruik word, later deur die reduksie van
asetaldehied na etanol regenereer word. Die nasending van die metaboliese vloeiing
van piruvaat na biomassavorming lei egter na ‘n oormaat NADH-vorming. Onder hierdie
toestande word die intrasellulêre redoksbalans dan hoofsaaklik deur die vorming van
gliserol onderhou. Laasgenoemde word veral deur twee ensieme beheer, naamlik
Gpd1p en Gpd2p. Die delesie van die gene wat vir hierdie twee proteïene enkodeer, lei
tot ‘n akkumulasie van NADH en veroorsaak dat die selle nie hulle gistingsvermoë en
groei onder anaërobiese toestande kan onderhou nie.
Die doelwit van hierdie studie was om die groei, gistingsvermoë en metabolietsintese
van verskeie gpd1Δgpd2Δ dubbelmutant (DM) rasse te ondersoek waarin die
redoksbalanseringspotensiaal gedeeltelik herstel is deur die uitdrukking van inheemse
of heteroloë gene. Rasse is gekonstrueer deur alternatiewe NADH-oksiderende weë in
te voer of deur bestaande weë te manipuleer om die oksidasie van oormaat NADH te
bevoordeel. Meer spesifiek het die modifikasies die volgende ingesluit: (i)
sorbitolvorming; (ii) die vestiging van ‘n weg vir propaan-1,2-diol-vorming; en (iii) die
verhoging van etanolvorming. Buiten die genetiese manipulering van die gpd1Δgpd2Δ
dubbelmutant, is die byvoeging van piruvaat tydens groei ook ondersoek. Die
eksperimente is onder suurstofbeperkte toestande in ‘n hoë-suiker medium uitgevoer en
die gegiste produk is ondersoek vir totale suikerverbruik, biomassa en primêre en
sekondêre metaboliete wat deur die verskillende rasse gevorm is. Die verhoudings
tussen suikerverbruik, groei en metabolietproduksie deur die verskillende rasse is
ondersoek deur die data wat deur die verskillende rasse gegeneer is deur middel van
meerveranderlike data-analise te vergelyk. Analise van die weë wat in die produksie
van primêre (sure, etanol en ander metaboliete) en sekondêre metaboliete (aromaverbindings) betrokke is, is ook uitgevoer om die verandering in vloei te bepaal in
vergelyking met die wildetipe (WT) ras.
Die resultate het gewys dat hierdie manipulasies die gistingsvermoë van die
gpd1Δgpd2Δ-dubbelmutant verbeter het, wat ‘n gedeeltelike herstel van NAD+-
regenerasievermoë voorstel, hoewel nie tot dieselfde mate as in die WT-ras nie. Soos
verwag, is ‘n beduidende korrelasie tussen suikerverbruik en etanol- en
biomassavorming gevind. Etanolopbrengs is deur genetiese manipulasies verhoog,
maar nie die finale konsentrasies van etanol nie. Sorbitol is in beduidende hoeveelhede
deur die DM(srlD) en DM(SOR1)-rasse gevorm en propaan-1,2-diol deur die DM(gldA,
GRE3, mgsA) -rasse, hoewel teen laer molare opbrengste as gliserol. Verder is die
opbrengs van sekondêre metaboliete (isobutanol, iso-amielalkohol, 2-fenieletanol en
isobottersuur) deur genetiese manipulasie verhoog, terwyl die etielasetaatkonsentrasie
en -opbreng verlaag is. Die resultate dui aan dat die aromaverbindingseienskappe van
wyngiste voordelig verander kan word deur die gliserolsintetiseringsweg te manipuleer.
Die byvoeging van piruvaat tydens die groei van die gpd1Δgpd2Δ-dubbelmutant dra by
tot uitermate NADH-reoksidasie tydens die bykomende vorming van etanol.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/3983 |
| Date | 03 1900 |
| Creators | Jain, Vishist Kumar |
| Contributors | Bauer, Florian, Prior, Bernard, University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 190 p. : ill. |
| Rights | University of Stellenbosch |
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