Generating lower ethanol yields in fermentations by Saccharomyces cerevisiae via diversion of carbon flux towards the production of fructo-oligosaccharides

Brandt, Bianca Anina (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79875
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