Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: There is a growing international consumer demand for the production of lower ethanol wines.
This can be attributed to various qualitative, social, economic and health concerns that are
associated with high ethanol wines (Kutyna et al., 2010; Varela et al., 2012). There is
continuous development and research into methods and technologies to lower the ethanol
concentration in wine. However, in addition to the added cost and complexity these technologies
all have various shortcomings. The development of yeast strains with lower ethanol productivity,
yet desirable organoleptic and fermentation capacity, therefore remains a highly sought after
research and development target in the wine industry.
Biologically based approaches aim to generate yeast strains with the capacity to divert
carbon from ethanol production towards targeted metabolic endpoints (Kutyna et al., 2010). This
should ultimately be achieved without the production of unwanted metabolites that can
negatively affect wine characteristics. In the context of these challenges, this study aimed to
investigate the use of fructans as carbon sinks during fermentation to divert fructose from
glycolysis and ethanol production toward intracellular fructan production by generating levan
producing strains. In addition, the impact of fructan production on metabolic carbon flux during
fermentation by these strains was analyzed. This was the first attempt to analyze intracellular
fructan production in Saccharomyces cerevisiae under fermentative conditions with fructans
acting as carbon sinks.
Fructans are fructose polymers that act as storage molecules in certain plants and
function as part of the extracellular matrix in microbial biofilms, and are intensively studied due
to their economic interest. Here we undertook the heterologous expression of a levansucrase
(LS) M1FT from Leuconostoc mesenteroides, an enzyme producing β(2-6) levan-type fructans,
in the S. cerevisiae BY4742Δsuc2 strains without invertase activity (encoded by SUC2).
Levansucrases indeed utilize sucrose as both fructose donor and initial polymerization
substrate, and the sucrose concentration is of import to maintain transfructosylation activity of
enzyme. High intracellular sucrose accumulation was achieved by the heterologous expression
of either a sucrose synthase (Susy; cloned from potato) or by growing strains expressing the
spinach sucrose transporter (SUT) in sucrose containing media. Endogenous sucrose synthesis
was of specific interest to the overall goal of the project, which was to reroute carbon flux away
from glycolysis in grape must containing only hexoses as carbon source. In addition, this
approach of combining intracellular sucrose production with intracellular levan production could
be used in various applications to limit the need for sucrose in media as both carbon source and LS substrate. The extracellular LS M1FT was introduced into Susy and SUT strains as either the complete
gene (M1FT) or 50bp truncation (M1FTΔsp) without the predicted signal peptide. The data show
that intracellular levan accumulation occurred in aerobic, but not anaerobic conditions. The data
also suggest that the production of levan did not impact negatively on general yeast physiology
or metabolism in these conditions. However, no significant reduction in ethanol yields were
observed, suggesting that further optimisation of the expression system is required. This is the
first report of levan synthesis by S. cerevisiae, and contributes towards expanding the
possibilities for further industrial applications of these compounds. / AFRIKAANSE OPSOMMING: Daar is toenemende aanvraag deur wynverbruikers na laër alkohol wyne. Hierdie neiging kan
toegeskryf word aan verskeie kwalitatiewe, gesondheids en sosio-ekonomiese redes wat
geassosieer word met die verbruik van hoër alkohol wyne. Daar is ’n deurlopende navorsing
dryf toegespits op metodes en tegnologieë om die alkohol konsentrasie van wyne te verlaag.
Hierdie tegnologieë het egter, bykomstig tot koste en kompliksiteits toename, verkeie
tekortkominge. Die ontwikkeling van gisrasse met verlaagde alcohol produksie, maar steeds
wenslike organoleptiese en fermentasie eienskappe, bly ‘n baie gesogte navorsings en
ontwikkeling teiken in die internasionale wyn industrie. Biologiese benaderings streef om gisrasse te genereer met die vermoë om koolstof weg
van etanol produksie te herlei na geteikende metabolise eindpunte. Hierdie doelwit moet ook
uiteindelik bereik word sonder die produksie van ongewenste metaboliete wat die wyn negatief
kan affekteer. In die konteks van hierdie uitdaging, het hierdie studie gestreef om die gebruik
van fruktane as ’n koolstof poel tydens fermentasie, met die doel om fruktose te herlei vanaf
glikolise en etanol produksie na intrasellulêre fruktane produksie. Om hierdie doelwit te bereik,
is gisrasse ontwikkel wat levaan (’n spesifieke fruktaan) produseer. Die impak van fruktaan
produksie op metaboliese koolstof vloei tydens fermentasie deur hierdie gisrasse is bykomsrig
ontleed. Hierdie verslag beskryf die eerste poging om intraselullêre fruktaan produksie in
Saccharomyces cerevisiae te bewerkstellig, met die doel om fruktaan as ’n koolstof poel te
gebruik. Fruktane is fruktose polimere wat as bergings molekules optree in sekere plante en ook
funksioneer as deel van die ekstrasellulêre matriks in mikrobiese biofilms. Hierdie polimere word
tans internasionaal intensief bestudeer weens hul ekonomiese belang. Hierdie studie beskryf
die uitdrukking van die levaansukrase (LS) M1FT van Leuconostoc mesenteroides, wat β(2-6)
levaan-tipe fruktane produseer, in S. cerevisiae BY4742Δsuc2 rasse, sonder invertase
(gekodeer deur SUC2). Levaansukrases gebruik inderdaad sukrose as beide ’n fruktose donor
en ook as ’n aanvanklike polimeriserings substraat. Die fruktose konsentrasie is belangrik om
transfruktosilerings aktiwiteit van die ensiem te handhaaf. Hoë intrasellulêre sukrose
akkumulasie was bereik deur die heteroloë uitdrukking van ’n sukrose sintase (Susy; gekloneer
van aartappel) of die spinasie sukrose transporter (SUT) in media bevattende sukrose.
Endogene sukrose sintese was van spesifieke belang tot die algehele doelwit om koolstof te
herlei, weg van glikolise tydens fermentase van druiwe sap. Die benadering om intraselullêre
sukrose produksie met levaan produksie te koppel, kan ook gebruik word in verskeie
toepassings om die afhanklikheid op sukrose in die media, as substraat vir LS, te verminder. Die ekstraselullêre LS, M1FT, was as vollengte geen (M1FT) of as ’n 50bp afkapping
(M1FTΔsp), sonder seinpeptied, in die Susy en SUT gisrasse uitgedruk. Die data dui aan dat
die produksie van levaan nie ’n negatiewe impak het op gis fisiologie of metabolisme in die toets
kondisies nie. Daar was egter geen waarbeenbare afname in etanol opbrengs nie, wat aandui
dat verdere optimisering van ekspressie sisiteem benodig word. Hierdie is die eerste verslag
van levaan sintese in S. cerevisiae en dra by tot die uitbreiding van moontlikhede vir indutriële
toepassings van die die verbindings. / IWBT and NRF
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79875 |
| Date | 03 1900 |
| Creators | Brandt, Bianca Anina |
| Contributors | Bauer, F. F., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xi, 63 p. : ill. |
| Rights | Stellenbosch University |
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