Dissertations / Theses on the topic 'Saccharomyces cerevisiae – Biotechnology'

Modelling studies were performed on a fermentation system using Saccharomyces cerevisiae NCYC 754. The production of fermentation product and cytochrome P-450 were studied under semi-anaerobic condition in batch cultures. The fermentation was carried out in a 5-litre fermenter and controlled at constant set-points which had been optimized by an earlier worker with respect to enzyme yield. An unstructured model was established to describe the biomass profile which comprised two growth phases; however the system did not demonstrate the classical diauxic growth as expected. Furthermore, against the general belief that glucose is the limiting substrate of the system; the maximum wet biomass seemed to depend on the concentration of peptone and yeast extract in the fermentation broth. Growth kinetics indicated that a second substrate was utilized before glucose metabolism began in spite of the presence of high levels of glucose. Luedeking and Piret type models, combined with ethanol inhibition, were derived to describe the profile of ethanol and cytochrome P-450 concentration. Later, it was demonstrated that a close correlation exists between initial glucose and cytochrome P-450 concentration. Viable count by agar plating techniques was used to test the proposed biomass model. The results were in line with the proposed model, even though the cell viability profile in the system was rather low. The Taguchi method was used to seek out the noise factor in the system, and optimize the operating conditions for a particular performance statistic. Contrary to earlier findings, the stirrer speed was found to have little effect on the yield of cytochrome P-450.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications.
AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006.
Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important quality factors, but these can be influenced by many factors, such as grape-derived compounds that exist as free volatiles and also as glycosidically bound. The chemical composition of wine is determined by factors such as grape variety, geographic position, viticulture condition, microbial ecology of the grape and the winemaking process. The varietals aroma is determined by both the volatile and the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene derivatives, which are naturally present in the wine. Monoterpenes are very important in the flavour and aroma of grapes and wine. They can be found in grapes and wine either in the free, volatile and odorous form, or in the glycosidically-bound, non-volatile and non-odorous form. The ratio of glycosidically-bound compounds to free aroma compounds is very high in the Gewürztraminer, Muscat and Riesling cultivars in particular. The glycosidic bonds can be hydrolysed either by the acid method or by using enzymes. The acid method is disadvantageous because it can modify the monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying the aroma character. The enzyme method of breaking the glycosidic bonds occurs in two successive steps: initial separation of glucose from the terminal sugar by a hydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase, depending on the aglycone moiety), followed by the breaking of the bond between the aglycone and glucose by β-glucosidase. The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera), bacterial, yeast or fungal sources. Most of the enzymes produced by these sources are not functional under the winemaking conditions of low pH, low temperature, high glucose and high ethanol content. However, β-glucosidases from fungal origins, particularly from Aspergillus spp., are tolerant of winemaking conditions. The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii (BGLA), which is linked to the cell wall and the free β-glucosidase, was to determine if anchoring the enzyme to the cell wall will increase the activity of the enzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG 97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmids pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1 anchor domains. All the plasmids were genome-integrated and expressed in the reference strain Saccharomyces cerevisiae 303-1A. All the transformants were grown in 2% cellobiose and showed higher biomass production compared to the reference strain. β-Glucosidase activity was also assayed and transformed strain W16 showed a fourfold increase in activity compared to the reference strain. There was no significant increase in the activity of the other transformed strains, W24, W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was 40ºC. The recombinant strains together with the reference strain were used to make wine from Gewürztraminer grapes. The levels of numerous monoterpenes were enhanced in the resultant wines. The concentration of nerol was increased fourfold, that of citronellol twofold, and geraniol was 20% higher than in the wild type. There was also an increase in the levels of linalool and a-terpinol, but this was not significant. In wines produced with W97, W98 and W24, monoterpene levels did not show a significant difference. In future, the expression of the W16 expression cassette in an industrial wine yeast strain could be performed. In combination with the production of enzymes such as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which are involved in the first step of enzymatic hydrolysis, this wine strain could release the bound monoterpenes and enhance the aroma of the wine.
AFRIKAANSE OPSOMMING: Wyn is ‘n komplekse medium. Wynaroma, -geur en -kleur is belangrike kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan wees of glikosidies gebind is. Die chemiese samestelling van wyn word bepaal deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande, mikrobiese ekologie van die druif en die wynbereidingsproses. Die variëteitsaroma word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene, norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom. Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn. Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in glikosidiesgebinde, nie-vlugtige en nie-geurige vorms. Die verhouding van glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die Gewürztraminer-, Muscat- en Riesling-kultivars. Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode gehidroliseer word. Die nadeel van die suurmetode is dat dit monoterpene kan modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter modifiseer nie. Die ensiemmetode waarmee die glikosidiese verbinding afgebreek word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose van die terminale suiker deur ‘n hidrolase (a-L-arabinofuranosidase, a-Lramnosidase of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur die verbreking van die verbinding tussen die aglikoon en glukose deur β- glukosidase. Die β-glukosidase-ensiem kan vanaf ‘n verskeidenheid plant- (Vitis vinifera), bakterie-, gis- en swambronne verkry word. Die meerderheid van die ensieme wat deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie. β- Glukosidase vanaf ‘n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter wynbereidingstoestande verdra. Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand geanker is. Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie studie gebruik. BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is gekoppel aan CWP2, en in pDLG 98 is dit aan AGa1-ankergebiede gekoppel. Al die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A genoomgeïntegreer en uitgedruk. Al die transformante is in 2% sellobiose gegroei en het hoër biomassaproduksie as die verwysingsras getoon. β-Glukosidaseaktiwiteit is ook geëssaieer en die getransformeerde ras W16 het ‘n viervoudige verhoging in aktiwiteit in vergelyking met die verwysingsras getoon. Daar was geen noemenswaardige verhoging in die aktiwiteit van die ander getransformeerde rasse, W24, W97 en W98, nie. Ensimatiese karakterisering vir optimum-pH en - temperatuur is gedoen – vir al die rasse was die optimum-pH 4 en die optimumtemperatuur 40ºC. Die rekombinante rasse, tesame met die verwysingsras, is gebruik om wyn met Gewürtztraminer-druiwe te maak. Die vlakke van talryke monoterpene is in die gevolglike wyne verhoog. Die konsentrasie van nerol is viervoudig verhoog, dié van sitronellol tweevoudig, en geraniol was 20% hoër as in die wilde tipe. Daar was ook ‘n verhoging in die vlakke van linaloöl en a-terpinol, maar hierdie verhoging was nie noemenswaardig nie. In wyne wat met W97, W98 en W24 gemaak is, het die monoterpeenvlakke nie ‘n noemenswaardige verskil getoon nie. In die toekoms sal die uitdrukking van die W16-uitdrukkingskasset in ‘n industriële wyngisras uitgevoer kan word. In kombinasie met die produksie van ensieme soos a-arabinofuranosidase, a-ramnosidase, β-apiosidase, wat in die eerste stap van ensimatiese hidrolise betrokke is, sal hierdie wyngisras die gebonde monoterpene kan vrylaat en die aroma van die wyn kan verbeter.

Thesis (MSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive bacterial cell walls, resulting in cell death. It is thus active against lactic acid bacteria (LAB), which may be present in grape juices and musts. These bacteria are responsible for malolactic fermentation of wines although many species, particularly of the genera Lactobacillus and Pediococcus, are considered spoilage organisms. The growth of LAB is therefore closely monitored and controlled during winemaking. The most common means of control is growth inhibition by chemical treatment (usually with SO2). Lysozyme is a commonly used wine processing aid, complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2 to be used, thus improving the wholesomeness of wine. The OIV (Organisation Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per liter of wine in 1997. This study evaluated the effect of different secretion signals on the secretion of lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate kinase 1) prompter and terminator was also evaluated. In the case of FY23 four secretion signals were used, namely the native lysozyme signal and the S. cerevisiae mating factor-a signal as well as mutants of these signals. These mutants incorporated two additional arginines at the N-terminus of the signals immediately downstream of the terminal methionine. The effect of these mutations was to increase the positive charge of the secretion signal N-terminals. The secretion signal-lysozyme fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter and terminator. The resulting expression cassettes were cloned into integrating vectors YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to transform FY23 and VIN13. FY23 as well as VIN13 transformants were evaluated in an artificial medium designed to reflect the nutrient content of grape juice, with particular attention being paid to assiminable nitrogen. Three hexose concentrations were tested in order to determine the effect thereof on lysozyme secretion titer. Lysozyme secreted under all tested growth conditions was found to be too low for detection by either enzymatic assay or HPLC-FLD. For this reason secreted lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently, lysozyme concentrations in the concentrates was determined by means of the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was also performed. No significant differences were found between native or MF-a secretion signals and their mutated counterparts in terms of secretion titer or proteolytic maturation. Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases, with both an authentically processed and a larger form, in which the secretion signal was not completely removed, being present. Lysozyme secreted with the native signal appeared to be correctly processed in all cases. Secretion titer from high copy number episomal FY23 tranformants was similar to that of integrants containing a single copy of the gene. Sugar concentration affected lysozyme production, with higher quantities of the enzyme being secreted when higher initial sugar concentrations were used. Lysozyme titers were extremely low (< 0:25 mg/L) with all expression cassettes under all the tested conditions with both FY23 and VIN13. In the case of the VIN13’s a lower final biomass was found for the secretor strain tested in comparrison to the VIN13 wild-type.
AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë (MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling (gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn. Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23 is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF- a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale metionien. Die effek van hierdie mutasies was om die positiewe lading van die uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2, gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees. Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.

Thesis (MTech (Food Technology)--Cape Peninsula University of Technology, 2009
The primary aim of this study was to establish the effects of both cavitation, (20 KHZ), and heat (55°C or 60•C) on Saccharomyces cerevisiae GC210 (MATa lys2) suspended in physiological saline. Fluorescent flow cytometry was used to determine the effects of moist heat and acoustic cavitation on S. cerevisiae cells. Results from this study could be used as a guide for use by the food industry for the combined use of heat and sonication to disinfect various solutions contaminated with S. cerevisiae.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol.
AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities.
AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W) geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1 geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie van die geen sodoende in sommige rasse voorkom. Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens, asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk. Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van wyngiste te verbeter. Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme van regulering van toepassing op PGU1 te identifiseer. As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity.
AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

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.

Thesis (PhD)--Stellenbosch University, 2010.
ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production.
AFRIKAANSE OPSOMMING: Gewilde wynstyle word dikwels gemaak van volryp, goed ontwikkelde druiwe, gekarakteriseer deur intense aromas en smaakkomponente wat direk met spesifike kultivars geassosieer word. ’n Nadelige gevolg om druiwe te lank aan die wingerdstok te laat bly hang sodat meer intense geurkomponente kan ontwikkel, is die toename in suikerinhoud. Hierdie addisionele suiker lei tot wyne met hoër alkoholvlakke. Te hoë alkoholvlakke kan wyn ongebalanseerd laat voorkom en die smaak nadelig beïnvloed. Dit, tesame met gesondheidsredes en anti-sosiale gedrag wat gekoppel kan word aan die inname van te veel alkohol, strenger wetgewing aangaande dronkbestuur en die toename in belasting op wyne met ’n hoër alkoholinhoud, het aanleiding gegee tot ’n aanvraag vir wyn met ’n verlaagte alkoholinhoud, sonder dat aroma- en geurkomponente ingeboet word. Alhoewel daar sekere fisiese/gemeganiseerde prosesse beskikbaar is om die alkohol in wyn te verwyder of te verminder, is ’n nadeel dat hierdie prosesse baie duur en arbeidsintensief is, en dat dit deur sommige wynpuriste as te ingrypend in die ‘natuurlike’ proses van wynmaak beskou word. Sommige van hierdie alkoholverwyderingsprosesse kan ook die wyn se geur- en aromakomponente nadelig beïnvloed. As alternatief tot hierdie fisies-chemiese prosesse word wyngiste reg oor die wêreld deur verskillende navorsingsgroepe ontwikkel sodat van die druifsuikers nie na alkohol omgeskakel word nie, maar eerder ander metaboliete. Belowende navorsingsresultate in ’n voorafgaande studie het aanleiding gegee tot hierdie navorsingsprojek. In hierdie studie word daar klem gelê op die ontwikkeling, deur middel van genetiese manipulering, van ’n industriële wynras van Saccharomyces cerevisiae sodat dit in staat sal wees om glukose-oksidase (GOX; b-D-glukose:suurstof oksidoreduktase, EC 1.1.3.4) te produseer. GOX kan reeds b-D-glukose in die medium oksideer na glukoonsuur (GA), wat sodoende verhoed dat dit verder gemetaboliseer word via glukolise, en dit het tot gevolg dat ’n gedeelte van die beskikbare suiker nie omgeskakel word na alkohol nie. Die strukturele glukose-oksidase-geen (GOX) van die voedsel-gegradiëerde fungus, Aspergillus niger is gebruik tydens die konstruksie van drie kassette (GOX1, GOX2 en GOX2LOX). Binne hiedie geenkassette is A. niger se GOX-geen se transkripsieinisiëring en -terminering onafhanklik deur die fosfogliseraat-kinase-1-promotor (PGK1P) en termineerder (PGK1T ) bewerkstellig. Om uitskeiding van GOX uit die gis te bewerkstellig, is daar van die a-spesifieke gisferomoon-a-faktor (MFa1S) in GOX1 gebruik gemaak, en in GOX2, van A. niger se eie natuurlike sekresiesein. Hierdie geenkassette is binne-in die genoom van twee labaratoriumgisrasse van S. cerevisiae (BY4742 en S1278b) asook een industriële wyngisras (VIN13) geintegreer. ’n Addisionele integreringskasset (die sogenaamde GOX2LOX-kasset) is gemaak om die IME1-geen van S. cerevisiae te elimineer. Binne die GOX2LOXkasset is GOX2 aan ’n loxP-kasset gekoppel. Die nuwe wyngis VIN13-D1 is verkry deur ’n genomiese integrasie van GOX2LOX binne-in die IME1-lokus. Om die niesporulerende GOX-produserende wyngis VIN13-D2 te verkry, is VIN13-D1 gesporuleer, onderwerp aan mikromanipulasie en toegelaat om te herdiploidiseer. Ontledings het aangedui dat (i) GOX2 aanleiding gegee het tot hoër vlakke van ekstrasellulêre glukose-oksidase aktiwiteit in vergelyking met GOX1; en (ii) dat die vlakke van uitgeskeide biologies-aktiewe glukose-oksidase vir die wyngisrasse aansienlik hoër was. Dit het verdere kleinskaalse wynfermentasies geregverdig. Die getransformeerde wyngis VIN13-D1 is op eksperimentele skaal in die maak van rooi- en witwyn geëvalueer. Ontledings van hierdie eksperimentele wyne het daarop gedui dat glukose-oksidase deur die VIN13-D1-gisselle geproduseer en suksesvol uitgeskei tydens die wynmaakproses is, en dat VIN13-D1 die fermentasie gedomineer het en die alkoholiese gisting voltooi het. Resultate het egter ook aangedui dat die geproduseerde glukose-oksidase nie baie aktief was onder die wynmaaktoestande wat in hierdie eksperimentele wynmaakproses gegeld het nie, en gevolglik was daar nie ’n drastiese verlaging in die alkoholvlakke sigbaar toe VIN13-D1 se wyne met VIN13 se wyne vergelyk is nie. Wynmonsters is deur middel van Fourier-transformasie-mid-infrarooispektroskopie (FT-MIR) ontleed ten einde die chemiese samestelling te bepaal, en gaschromatografie-massaspektrometrie (GCMS) is aangewend om die wynaromakomponente te bepaal. Die vlakke van glukoonsuur is deur middel van ensiematiese reaksies bepaal. Multiveranderlike data-analise [hoofkomponentanalise (PCA) en parsiële kleinte kwadrate (PLS1) diskriminantanalise] is op die data van die verskeie analitiese tegnieke toegepas om onderliggende veskille tussen die wyne van VIN13 (wilde-tipe) en VIN13-D1 uit te wys. Chemometriese projeksies het aangetoon dat daar wel beduidende variasies sigbaar was tot en met drie hoofkomponente en/of PLS-komponente wat duidelik aandui dat glukoonsuur ’n sleutelfaktor was ten opsigte van die uitwerking wat GOX op VIN13-D1- fermentasies in vergelyking met VIN13-fermentasies. VIN13-D1 effek manifestasies is die beste waargeneem op grafieke wat PLS1-diskriminantanalise-data bevat. Verder het PLS1-diskriminantanalise ook aangetoon dat van die ‘groepe’ wat gebruik was tydens die analise, die FT-MIR-komponente en die GC-komponente beter resultate gelewer het. Die GC-komponente het hulle verder daartoe geleen om tussen die verskillende kultivars en VIN13/VIN13-D1-fermentasies te diskrimineer. Daar kan dus met sekerheid gesê word dat die grootste invloed in VIN13-D1 wyne sigbaar is in die aromakomponent, maar omdat daar wel ook variasies sigbaar was in die MIR-komponente, dat die smaakkomponente ook beïnvloed was. Die aktiwiteit van GOX in druiwesap is verder ondersoek deur gebruik te maak van kleinskaalse fermentasies in bioreaktors. Daar is bevestig dat die VIN13-D1- geproduseerde GOX biologies-aktief was tydens aerobiese kondisies, onaktief was tydens anaerobiese kondisies, en onmiddelik geaktiveer kon word wanneer ’n anaerobiese fermentasie aerobies gemaak word (gesimuleerde mikro-oksigenasie). Hierdie verskillende fermentasies dui daarop dat glukose-oksidase inderdaat aktief is in druiwesap, en dat suurstof ’n sleutelfaktor is tydens die aktivering van die ensiem. Met behulp van ’n vereenvoudigde model kon aangetoon word dat tydens ideale toestande dit wel moontlik is om die alkoholvlakke te verlaag na ’n voorafbepaalde konsentrasie vir die bereiding van lae-alkohol wyne. Hierdie studie verskaf verdere insig hoe om ’n GOX-produserende wyngis gedurende die wynmaakproses vir die verlaging van die alkoholvlakke te benut. Verder is dit duidelik dat suurstof van kardinale belang is vir die aktivering van die glukoseoksidase- ensiem en dat ’n tegniek soos mikro-oksigenasie ’n belangrike rol in hierdie verband tydens die wynmaakproses sou kon speel.

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O estabelecimento de condições para a realização de fermentações em meio sintético em escala de laboratório é uma pesquisa relevante que permite estudar alterações metabólicas e genéticas das leveduras durante os ciclos sucessivos de fermentação. A composição da matéria-prima industrial apresenta alterações constantes e os dados industriais são obtidos sem a precisão e reprodutibilidade dos dados obtidos em pesquisas científicas. Em uma seqüência longa de bateladas repetidas com o reuso de células, a recuperação das células mortas ou a simples perda celular da viabilidade, causadas por morte celular, ou a simples perda de viabilidade causada pelas condições do processo não podem ser ignoradas. Um excesso de biomassa no final do processo indica que a população de leveduras passou por renovação celular. Fermentações descontínuas alimentadas por pulsos decrescentes de sacarose foram utilizadas no presente trabalho. Como o inóculo usado foi muito alto, o crescimento celular foi menor durante a fermentação. O volume final de trabalho dentro do reator, contendo inicialmente todos os componentes exceto sacarose e leite de levedura, atingiu um aumento de 25% no seu depois de 4 horas de alimentação. Devido aos inóculos altos utilizados, um tratamento para recuperação do leite de levedura foi necessário entre os ciclos de fermentação a fim de minimizar as perdas em biomassa e viabilidade. Dados obtidos mostraram que uma redução no tamanho do inóculo levou a perdas em viabilidade. Depois da adição de cada pulso de sacarose, um reajuste do pH do meio para 4,5 foi necessário. Uma solução de água contendo sacarose e extrato de levedo nos permitiu revitalizar o leite de levedura com ganhos em biomassa e viabilidade durante 15 horas de incubação à 30ºC antes do ciclo de fermentação seguinte. Entre os ciclos de fermentação, perdas dramáticas...
The establishment of conditions to carry out fermentations in synthetic medium at laboratory scale is a very relevant research that will allow us to study metabolic and genetic alterations during the successive cycles of fermentation. The composition of the raw industrial materials show constant alterations and the industrial data are generally obtained without the precision and reproducibility of the data obtained in scientific research. In a long sequence of repeated batch processes with cell reuse, the recuperation of dead cells or the simple loss of viability caused programmed cell death or simply loss of viability caused by process conditions can not be ignored. Biomass excess at the end of the process indicates that the yeast population went through a cell renewal. Discontinuous fermentations fed by decreasing pulses of sucrose were used in the present work. As inoculum was very high, the growth was low during the fermentation. The volume inside the reactor, containing initially all medium components except sucrose and yeast cream, reached a 25% increase in its working volume after 5 hours feeding. Due to high inoculums used, a treatment for the yeast cream recuperation was required between the fermentation cycles in order to minimize the losses in biomass and viability. Data obtained showed that a decrease in the inoculums size leads to drops in viability. After the addition of each sucrose pulse, a pH readjustment to 4.5 was required. A water solution containing sucrose and yeast extract allowed us to revitalize the yeast cream with gains in biomass and viability during 15 hour incubation at 30ºC before the next fermentation cycle. Between the fermentation cycles, dramatic drops in viability were observed when an amount of 200 g.L-1 sucrose was added in relation to the final volume of medium inside the reactor, but only discrete variations were observed when 156 g.L-1 sucrose... (Complete abstract click electronic access below)

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A principal meta deste trabalho foi a de realizar estudos com leveduras industriais brasileiras utilizadas em usinas, na tentativa de viabilizar o emprego da Tecnologia de Fermentação de Mostos com Altos Teores de Açúcares Fermentecíveis, na produção de etanol combustível. Inicialmente, foram realizados experimentos para obtenção de informações relativas as linhagens, na fermentação de sacarose, maltose e glicose, em meios contendo uma base nitrogenada e suplementado com fontes de nitrogênio com diferentes complexidades estruturais. Os estudos mostraram que as linhagens industriais apresentam diferentes perfis de crescimento nos meios suplementados com diferentes fontes de nitrogênio, que variaram de um simples sal de amônio, a um hidrolisado ácido de proteínas (casaminoácidos) e hidrolisado enzimático de proteínas (peptona). Entretanto, a maior capacidade de acúmulo de biomassa pode não ter um reflexo direto na capacidade de utilização de açúcares e a consequente produção de etanol pelas leveduras industriais. Além da natureza estrutural da fonte de nitrogênio e do tipo de açúcar, a presença do oxigênio, em maior quantidade nos cultivos agitados, interferiu sobremaneira no desempenho fermentativo das leveduras industriais, e até no acúmulo de trealose. No geral, foi a suplementação com peptona que propiciou maior produção de biomassa, preservação da viabilidade celular e consumo mais eficiente da fonte de carbono, em cultivos agitados e não agitados. Os estudos com alta densidade celular tiveram como objetivo definir condições experimentais para a condução da Fermentação de Mosto com Altos Teores de Açúcares Fermentescíveis, e que pudessem propiciar ao final do processo a completa utilização da sacarose, associada á preservação da viabilidade...
In this work studies with Brazilian yeast for ethanol production were conducted in order to verify the possibility of utilization of brazilian yeasts for ethanol production in an industrial process known as Very High Gravity Sucrose Fermentation Technology, to produce wine with high ethanol contends. This technology has the advantage to reduce production costs by increasing fermentation yields. Initially, studies were conducted with sucrose, maltose and glucose fermetation in medium containing YNB, supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that yeast strains vary in their response to nitrogen source complex structure, kind of sugar and to oxygen availabity. In general, under peptone supplementation all strains, in shaking and static conditions, showed higher biomass accumulation, efficient sugar utilization and yeast viability was preserved. Sugar utilization by industrial strains not always was directly correlated with higher biomass accumulation. Trehalose accumulation was also influenced by the structural complexity of nitrogen sources, the kind of sugar and the presence of oxygen. Studies with high cell density were conducted to define experimental parameters for Very High Gravity Sucrose Fermentation, in order to induce at the end of process full sugar exhaustion together cell viability preservation. Complete sucrose utilization was detected only in media with 22 and 25% (w/v) sucrose. In the presence of higher sucrose contends (30% (w/v)), total sucrose exhaustion was obtained in a sugar cane based medium, supplemented with 2% (w/v) peptone. The results described in this thesis suggest that industrial yeasts show differing nitrogen demand, and the utilization of Very High Gravity Sucrose Fermentation technology could be carried only after finding the appropriated nutritional and fermentation conditions

The fuel ethanol industry is now making use of a very efficient process where virtually all sugar substrates are converted to ethanol. Nevertheless, some metabolic by-products excreted from Saccharomyces cerevisiae tend to reduce the ethanol yield. Of such, glycerol is the major one, accounting for about 5-10% relative to the amount of ethanol produced.
Glycerol plays an important role in maintaining the redox balance within the cells by oxidizing the cytosolic NADH under anaerobic conditions. It is also believed that it acts as an osmoprotectant and would be favourably produced in high osmotic pressure conditions.
In order to mitigate the production of glycerol, various aeration strategies were investigated in a single-stage continuous fermentation system. Oxygen dissolved in the fermentation medium put the yeast in aerobiosis, acted as an oxidizing agent and hence minimised the specific glycerol production by 36% as compared to a completely anaerobic fermentation.
This has hardly been reproduced in a more industrially relevant system using a multi-stage continuous fermentation process. Indeed, oscillations in the concentrations of the various metabolites over time made difficult the assessment of significant changes. Nevertheless, these findings open the door to further investigations in order to understand the effect of oxygen in continuous fermentations using very high gravity feeds, such as in the fuel ethanol industry.

Dissertation (PhD)--Stellenbosch University, 2007.
PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology.
AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC 3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek of dit op te neem en intrasellulêr te benut. Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/ transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen 0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te fermenteer. Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1 ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium. Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende anaerobe geassosieer word. Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut. Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg word.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009.
Saccharomyces cerevisiae has been used for millennia for the leavening of dough and in the production of alcoholic beverages such as beer and wine. More recently, it is being used as cell factories for the production of important pharmaceutical products. S. cerevisiae has also been extensively used as a model organism for studying many genetic and biochemical processes within the eukaryotic cell. Since the completion of a yeast genome sequence, many functional analysis projects have emerged with the aim of elucidating the functions of the unidentified genes revealed by the genome sequence. One of the most relevant approaches consisted in the construction of a collection of mutants deficient in all single genes, either in a haploid background for non-essential genes, or as heterozygous diploids for essential genes. This collection of strains can be subjected to phenotypic screens that might reveal the function of unknown genes or add to our understanding of already annotated genes. While this approach is promising, it also bears some limitations. For instance, many mutants have no overt phenotypes and some phenotypes do not obviously showcase the function of the encoded protein. In this study, S. cerevisiae strains with single deletions of genes involved in pyruvate metabolism were selected from the Euroscarf deletion library. Pyruvate is a central intermediate of glycolysis, and pyruvate metabolism largely defines the general distribution of carbon flux in the cell. These mutants were screened for modified fermentation kinetics or modified carbon flux under wine fermentative conditions, an environment that had not been previously used for the analysis of these mutants. A strain disrupted in the PDA1 gene, which encodes the E1α subunit of the pyruvate dehydrogenase showed a significant change in phenotype when grown in wine fermentative conditions. In particular, the mutant displayed a prolonged lag phase, but upon entering exponential growth, fermented significantly faster than the wild type strain and completed alcoholic fermentation in a shorter period of time. This phenotype could be of significant industrial interest. The mutant phenotype was further investigated through disruption of the gene in the same as well as in different genetic backgrounds, and through complementation of the PDA1 deletion with a plasmid-born wild type copy. The data show that the PDA1 gene disruption is not solely responsible for the observed phenotypes under wine fermentative conditions. We therefore propose that secondary mutations have contributed to the mutant phenotype. This study shows that phenotypes attributed to a specific gene in mutants of the Euroscarf library should always be confirmed before performing consequent experiments and drawing significant conclusions.

The chemical diversity and biological activities of terpene and terpenoids have served in the development of new flavors, fragrances, medicines and pesticides. While terpenes are made predominantly by plants and microbes in small amounts and as components of complex mixtures, chemical synthesis of terpenes remains technically challenging, costly and inefficient. In this dissertation, methods to create new yeast lines possessing a dispensable mevalonate biosynthetic pathway wherein carbon flux can be diverted to build any chemical class of terpene product are described. The ability of this line to generate diterpenes was next investigated. Using a 5.5 L fed bath fermentation system, about 569 mg/L kaurene and approximately 207 mg/L abietadiene plus 136 mg/L additional isomers were achieved. To engineer more highly modified diterpenes might have greater industrial, agricultural or medicinal applications, kaurenoic acid production reached 514 mg/L with byproduct kaurene and kaurenal at 71.7mg/L and 20.1mg/L, respectively, in fed batch fermentation conditions. Furthermore, ZXM lines for engineer monoterpene and ZXB lines for engineer triterpene were generated by additional specific genomic modification, 84.76 ±13.2 mg/L linalool, 20.54±3.8 mg/L nerolidol and 297.7mg/L squalene were accumulate in ZXM144 line ana ZXB line, respectively, in shake flask conditions.

CPF1 (centromere promoter factor 1) of Saccharomyces cerevisiae is a multifunctional protein. This multifunctionality may be associated with the different forms of the protein. To date, no clear function has been attributed to this protein. The general roles currently attributed to CPF1 are: maintenance of proper chromosome segregation or centromere function; methionine prototrophy and chromatin modulation. A series of recent papers have suggested the possibility of CPF1 being involved in protein-protein interactions. This thesis shows that CPF1 has an additional protein-protein interaction domain which is a coiled-coil. A truncation of CPF1 coding for only the C-terminal dimerization motif was cloned into a 17 expression vector and the protein, Dd, was produced in and purified from E. coli. Characterisation of the conformation of the C-terminal dimerization domain of CPF1 show the presence of a leucine zipper in Dd and that Dd forms a dimer under physiological conditions. Sequence alignment analysis of CPF1 and other bHLHZIP proteins identified a conserved serine residue in the basic domain of CPF1 and a conserved asparagine residue in the leucine zipper. Point mutations were introduced separately mutating these residues into either alanine or glutamate. The mutant alleles were cloned into yeast expression vectors. The cloned alleles were used to transform YAG93, a cpf1 deletion strain. Characterisation of the transformants obtained indicate the possibility of CPF1 being involved in the spindle assembly checkpoint control, possibly as a tethering protein. Finally, attempts were made to identify interacting partners of CPF1 and a purification scheme was designed to purify a putative CPF1 complex. Initially, a GST-CPF1 chimera was used to screen radioactively labelled crude yeast extracts for interacting partners of CPF1. This strategy identified five or six potential proteins which might form a multiprotein complex with CPFl. Attempts were then made to purify this putative complex from S. cerevisiae. The partially purified extracts were tested for CDEI activity, indicative of the presence of CPF1 in the samples and. tjhen analysed for protein content. The samples were also tested in a Holiday junction binding assay and in a histone acetyltransferase activity assay. In conclusion, CPF1 is a bHLHZIP protein that has a role in nutrient signalling and checkpoint control, possibly by acting as an intermediary protein that recruits essential factors to gene promoters or the centromere by protein-protein interactions.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour.
AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae neem veranderinge in sy omgewing waar en reageer daarop deur middel van spesifieke sellulêre programme, in die besonder deur geenuitdrukking aan te pas. Verskeie aanpasbare veranderinge beïnvloed die fisieke, asook chemiese eienskappe van die selwand, en talle meganismes is al beskryf wat die uitdrukkingsvlakke beïnvloed van gene wat vir selwandkomponente kodeer. Die modifikasie van die selwand het ’n direkte impak op selwand-eienskappe, asook die sel-sel- en sel-oppervlak-interaksies. Verskeie van hierdie eienskappe word direk gekoppel aan die selwandproteïenfamilies, wat ook as adhesie-faktore bekend staan. Veral lede van die Flokkulasie (FLO) -geenfamilie het ’n noodsaaklike funksie in adhesie-fenotipes. Flo11p speel ’n rol in verskeie fenotipes, wat insluit die indringende groei van agar, plastiekaanhegting en die vorming van pseudohifes, “flor“ en “matte“, terwyl Flo1p flokkulasie beheer. Die regulering van FLO11-uitdrukking is deeglik gedokumenteerd en dit word hoofsaaklik gereguleer deur die mitogeen-geaktiveerde proteïenkinase (MAPK) en sikliese AMP-proteïenkinase A (cAMP-PKA) seintransduksiekaskades. Genetiese analises toon dat Mss11p stroom-af en sentraal tot hierdie kaskades funksioneer, en dit aktiveer transkripsie deur interaksie met die cAMP-PKA-komponent, Flop8. In hierdie studie word ’n ondersoek gedoen na addisionele teikengene van Flo8p en Mss11p, en hoe hierdie gene gereguleer word, asook hul impak op selwandeienskappe en geassosieerde adhesie-fenotipes. Ons analises toon dat Mss11p ook benodig word vir die ekspressie van FLO1 en dat dit, tesame met Flo8p, beheer uit oefen oor verskeie Flo-afhanklike fenotipes. Genoomwye geenekspressie-analises wys verder daarop dat veranderde Mss11p-vlakke lei tot die aanpassing van die ekspressie van verskeie selmembraan- en selwandgene, naamlik AQY2 asook lede van die DAN- en TIR-geenfamilies. Verdere genetiese analise dui daarop dat adhesie-fenotipes byna eksklusief afhanklik is van FLO-geenekspressie. Daar is verder getoon dat hierdie fenotipes ook Flo10p benodig en dus afhanklik is van die spesifieke balans van Floproteïene in die selwand. Die analise van seintransduksiemutante demonstreer dat FLO10 en FLO11 seintransduksie-komponente deel, maar dat hierdie gene verskillend gereguleer word. Anders as FLO11, toon FLO10 nie ’n absolute noodsaaklikheid vir Mss11p nie, maar eerder vir die MAPK-komponent, Ste12p. Totale genoomekspressie-analises is ook gedoen op gisrasse met aangepaste vlakke van Flo8p en dis vergelyk met bogenoemde transkripsiedatastel. Hierdie analise wys dat Flo8p and Mss11p die FLO-gene, asook AQY2 en TIR3, koreguleer, maar ook beduidende unieke teikengene het. Die kombinasie van transkripsiedata met huidig beskikbare informasie betreffende transkripsiefaktor (TF) -interaksienetwerke dui op die relevansie van netwerkinteraksie tussen Cin5p, Flo8p, Mga1p en Mss11p. Hiervan is daar ’n model opgestel waarin Flo8p in die meeste gevalle as die aktiverende TF-komponent optree, terwyl Mss11p as TF-teiken dien, moontlik as ’n mediator- of polimerase II holoënsiemkomponent. Laatens word daar vir die eerste keer verslag gedoen van ”mat”-vorming deur ’n industriële wyngisras en toon ons verder dat hierdie fenotipe beduidend verander word deur middel van die aanpassing van FLO11-uitdrukking.

Selenium (Se) is an essential element. Supplementation of Se as yeast-Se in animal and human diets has been proven to have beneficial health effects. The goal of this study was to add a maximum amount of Se in yeast metabolism in order to optimize its incorporation in amino acids. A bakery yeast strain of Saccharomyces cerevisiae (S. cerevisiae) was studied for its tolerance to Se when the latter was incorporated at different levels. Fermentations were run at 27°C for 8 h and 24 h. The maximum Se incorporation was achieved when 12.6 mmol of Se, as sodium selenite salt, was added to the culture medium and fermented for 24 h. A final Se concentration of 1550 +/- 35 mug/g yeast was obtained by this treatment.
Different yeast strains of S. cerevisiae were also studied for their capacity to incorporate Se. Five yeast strains of wine and four yeast strains of beer were fermented for 24 h and tested for their capacity to incorporate Se. The amount of 12.6 mmol Se was added in the growth medium. A maximum of 642.6 +/- 3.6 mug Se/g yeast was found to be incorporated in Uvaferm BC wine strain. Uvaferm windsor of beer strain was able to incorporate a maximum of 826.8 +/- 10.4 mug Se/g yeast. These yeast strains could be used as alternatives for Se supplementation.
Se speciation was carried out on the bakery yeast strain containing 1550 +/- 35 mug Se/g yeast, using Fast Phase Liquid Chromatography (FPLC) and amino acid analysis. Out of 1550 +/- 35 mug Se/g yeast, 57.5% Se was present as selenoaminoacids. The yeast extract contained 147 +/- 14 mug/g of SeCys, 248 +/- 13 mug/g of SeCyst and 295 +/- 17 mug/g of SeMet. Yeast cell walls contained 65 +/- 8 mug/g of SeMet; 69 +/- 5 mug/g of SeCyst and 67 +/- 9 mug/g of SeCys. These selenoaminoacids are known for their beneficial health effects. The produced Se-enriched bakery yeast could be used, after evaluated to be toxicologically safe, as an efficient dietary supplement.

Resumo: O estabelecimento de condições para a realização de fermentações em meio sintético em escala de laboratório é uma pesquisa relevante que permite estudar alterações metabólicas e genéticas das leveduras durante os ciclos sucessivos de fermentação. A composição da matéria-prima industrial apresenta alterações constantes e os dados industriais são obtidos sem a precisão e reprodutibilidade dos dados obtidos em pesquisas científicas. Em uma seqüência longa de bateladas repetidas com o reuso de células, a recuperação das células mortas ou a simples perda celular da viabilidade, causadas por morte celular, ou a simples perda de viabilidade causada pelas condições do processo não podem ser ignoradas. Um excesso de biomassa no final do processo indica que a população de leveduras passou por renovação celular. Fermentações descontínuas alimentadas por pulsos decrescentes de sacarose foram utilizadas no presente trabalho. Como o inóculo usado foi muito alto, o crescimento celular foi menor durante a fermentação. O volume final de trabalho dentro do reator, contendo inicialmente todos os componentes exceto sacarose e leite de levedura, atingiu um aumento de 25% no seu depois de 4 horas de alimentação. Devido aos inóculos altos utilizados, um tratamento para recuperação do leite de levedura foi necessário entre os ciclos de fermentação a fim de minimizar as perdas em biomassa e viabilidade. Dados obtidos mostraram que uma redução no tamanho do inóculo levou a perdas em viabilidade. Depois da adição de cada pulso de sacarose, um reajuste do pH do meio para 4,5 foi necessário. Uma solução de água contendo sacarose e extrato de levedo nos permitiu revitalizar o leite de levedura com ganhos em biomassa e viabilidade durante 15 horas de incubação à 30ºC antes do ciclo de fermentação seguinte. Entre os ciclos de fermentação, perdas dramáticas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The establishment of conditions to carry out fermentations in synthetic medium at laboratory scale is a very relevant research that will allow us to study metabolic and genetic alterations during the successive cycles of fermentation. The composition of the raw industrial materials show constant alterations and the industrial data are generally obtained without the precision and reproducibility of the data obtained in scientific research. In a long sequence of repeated batch processes with cell reuse, the recuperation of dead cells or the simple loss of viability caused programmed cell death or simply loss of viability caused by process conditions can not be ignored. Biomass excess at the end of the process indicates that the yeast population went through a cell renewal. Discontinuous fermentations fed by decreasing pulses of sucrose were used in the present work. As inoculum was very high, the growth was low during the fermentation. The volume inside the reactor, containing initially all medium components except sucrose and yeast cream, reached a 25% increase in its working volume after 5 hours feeding. Due to high inoculums used, a treatment for the yeast cream recuperation was required between the fermentation cycles in order to minimize the losses in biomass and viability. Data obtained showed that a decrease in the inoculums size leads to drops in viability. After the addition of each sucrose pulse, a pH readjustment to 4.5 was required. A water solution containing sucrose and yeast extract allowed us to revitalize the yeast cream with gains in biomass and viability during 15 hour incubation at 30ºC before the next fermentation cycle. Between the fermentation cycles, dramatic drops in viability were observed when an amount of 200 g.L-1 sucrose was added in relation to the final volume of medium inside the reactor, but only discrete variations were observed when 156 g.L-1 sucrose... (Complete abstract click electronic access below)
Orientador: Cecília Laluce
Coorientador: Reinaldo Marchetto
Banca: Maria Lúcia Gonsales da Costa Araujo
Banca: Daniel Ibraim Pires Atala
Mestre

An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. Far11, a component of a six-member complex, was found to interact with Tpd3 and Pph21, conserved components of Protein Phosphatase 2A (PP2A) via co-immunoprecipitation. Mutations in FAR11 and RTS1, which encodes a PP2A regulatory B subunit, restore phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. These data suggest that TORC2 signaling is antagonized by Far11-dependent PP2A activity. To characterize the assembly of the Far complex in vivo, intracellular localization of the Far complex was examined by fluorescence microscopy. It was found that the Far complex localizes to the endoplasmic reticulum (ER). The data show that Far9 and Far10 are tail-anchored proteins that localize to the ER first and recruit a Far8-Far7-Far3 pre-complex. Far11 is found at the ER only when all other Far proteins are assembled at the ER. Surprisingly, ER localization is required for the Far Complex’s role TORC2 signaling because deletion of the tail-anchor domain of Far9 results in partial bypass of the tor2-21 mutant growth defect at 37 ˚C.

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

Thesis (MSc (Microbiology))--Stellenbosch University, 2011.
ENGLISH ABSTRACT: Reliable energy resources could be considered as one of the cornerstones of the prosperity of the human race. The growing human population is constantly exerting more pressure on the world’s natural resources, which include natural fossil fuels that are non‐renewable. There are concerns regarding the use of fossil fuels due to its growing scarcity and its negative impact on the environment. There is thus a growing need in the world for energy sources that are renewable, more or less carbon neutral and therefore with a minimum environmental impact. Renewable energy is currently being harnessed from the wind, water and sun, but to a limited extent. These forms of natural resources are very attractive for the production of renewable energy, but these technologies are difficult to apply in the current transportation sector. Biofuels provide an alternative to the current use of liquid fossil fuels and it could be able to sustain the current fleet of automobiles worldwide in the intermediate to long term with minimal adjustment to the engines of these vehicles. Extensive research has been done on the production processes for biofuels. Previous processes included the use of high temperatures and acids that further increased the total production cost and thus making biofuels less attractive as an alternative energy source. Recent research has suggested a wide range of organic materials as substrate for the production of biofuels, which include lignin, hemi‐cellulose, cellulose and starch. Processes based on hemi‐cellulose, cellulose and lignin as substrate are still in its early research stages and commercial application of these processes will only occur over the medium‐ to long‐term. Starch is a very good alternative source for the production of biofuels, but there is a need for a microbial system for the conversion of starch to bio‐ethanol in a single step, referred to as Consolidated Bioprocessing (CBP). This would reduce the overall production cost of bio‐ethanol and thus making starch‐based substrates more attractive as an alternative energy source. The cost saving will be mainly due to the elimination of the pre‐treatment of raw starch at high temperatures and the addition of enzymes for the liquefaction and saccharification of starch to simple sugars. However, as there is no currently no known microbial organism known that can produce the required enzymes (i.e. amylases) as well as ferment the resulting sugars to ethanol, heterologous expression of these enzymes in a host strain able to ferment sugars could provide the best alternative system. In the first part of this study, 36 fungal strains known for the production of amylases were screened and compared for the highest extracellular enzyme activity on raw corn starch. The best two candidates, i.e. Aspergillus tubingensis (T8.4) and Mucor cincinelloides (1180), were then further evaluated to determine which organism has the highest efficiency when combined with a Saccharomyces cerevisiae laboratory strain. In fermentation experiments, A. tubingensis (T8.4) in combination with S. cerevisiae Y102 yeast strain resulted in the highest yield of ethanol. Literature on A. tubingensis is limited compared with other Aspergillii and it was previously accepted that A. tubingensis has the highest homology with Aspergillus niger. However, other reports – including the present study ‐ found that A. tubingensis is closer related to other Aspergillus spp. with regard to its amylolytic enzymes. The α‐amylase gene of A. tubingenis has a homology of 99.00% with that of Aspergillus kawachii whereas the glucoamylase gene has a homology of 99.26% with that of Aspergillus shirousami. In the second part of this study, two recombinant S. cerevisiae strains were constructed to express the wild type A. tubingensis α‐amylase (Atamy) and glucoamylase (Atglu), respectively. The combination of the two recombinant yeast strains was able to completely hydrolyse and also utilize raw corn starch for the production of bio‐ethanol, with a yield of 11.04 g/l of ethanol, which translates to 98% of the theoretical yield from starch with a 52% conversion of the total raw starch. This rate of conversion is lower than other reports which indicated up to 82% and 96% of the theoretical yield of ethanol from raw and soluble starch, respectively, by α‐ and glucoamylase. Furthermore, the combined expressed of the two genes was much more effective than when only one of the two genes were expressed, with a yield of 0.32 g/l ethanol for only Atamy and 2.52 g/l ethanol for Atglu. This proved that the combination of the A. tubingensis genes were best suited for the production of biofuels from raw starch. This also proved that the concept of constructing an amylolytic yeast strain capable of raw starch hydrolysis and fermentation was indeed feasible.
AFRIKAANSE OPSOMMING: Betroubare energiebronne kan as een van die boublokke vir die vooruitgang van die mensdom beskou word. Die groeiende menslike populasie is gedurig besig om meer druk op die wêreld se natuurlike hulpbronne te plaas, insluitende nie‐hernubare fossielbrandstowwe. Daar is kommer rakende die gebruik van fossielbrandstowwe weens ‘n afname in die beskikbaarheid en die negatiewe impak wat dit op die omgewing het. Daar is dus ‘n groeiende behoefte in die wêreld vir ‘n hernubare, min of meer koolstof‐neutrale energiebron wat ‘n minimale omgewingsimpak sal hê. Hernubare energie word tans tot ‘n beperkte mate uit wind, water en die son verkry. Hierdie vorms van natuurlike energie hulpbronne is baie aanloklik vir die vervaardiging van hernubare energie, maar hierdie tegnologië is moeilik toepasbaar in die huidige vervoersektor. Biobrandstowwe voorsien ‘n alternatief vir die huidige gebruik van fossielbrandstowwe en kan moontlik die huidige voertuigvloot wêreldwyd oor die medium‐ tot langtermyn onderhou met minimale enjinaanpassings van hierdie voertuie. Deeglike navorsing is alreeds op die vervaardigingsprosesse vir biobrandstowwe gedoen. Vorige prosesse het die gebruik van hoë temperature en sure ingesluit wat produksiekostes verder verhoog en gevolglik die gebruik van biobrandstowwe as ‘n alternatiewe energiebron minder aantreklik gemaak het. Onlangse navorsing het die gebruik van organiese materiaal as substraat vir die produksie van biobrandstowwe voorgestel, wat lignien, hemi‐sellulose, sellulose en stysel insluit. Prosesse met die gebruik van hemi‐sellulose, sellulose en lignien as substraat is nog in die beginfase van ontwikkeling en kommersialisering van hierdie prosesse sal eers oor die medium‐ tot langtermyn plaasvind. Stysel is ‘n baie goeie alternatiewe bron vir die produksie van biobrandstowwe, maar ‘n mikrobiese sisteem word vir die omskakeling van stysel in bio‐etanol in ‘n enkele stap benodig, bekend as gekonsolideerde bioprosessering (GBP). Dit sal die algemene produksiekoste van bio‐etanol verlaag en dus styselsubstrate as ‘n alternatiewe energiebron meer aantreklik maak. Die kostebesparing sal hoofsaaklik realiseer omdat die vooraf‐behandeling van rou stysel byhoë temperature en die toevoeging van ensieme vir die vervloeiing en versuikering van stysel tot eenvoudige suikers, uitgeskakel word. Aangesien daar tans geen bekende mikrobe organisme is wat die nodige ensieme (nl. amilases) kan produseer en ook die suikers wat daardeur vrygestel is, na etanol kan fermenteer nie, kan die heteroloë uitdrukking van hierdie ensieme in ‘n gasheer‐ras wat die suikers kan fermenteer, moontlik die beste alternatief verskaf. In die eerste deel van hierdie studie is 36 fungi rasse wat bekend is vir hul amilase produksie geevalueer en met mekaar vergelyk vir die hoogste ekstrasellulêre ensiemaktiwiteit op rou mieliestysel. Die beste twee kandidate, naamlik Aspergillus tubingensis en Mucor cincinelloides, is verder ge‐evalueer om te bepaal watter organisme het die hoogste effektiwiteit in kombinasie met ‘n Saccharomyces cerevisiae laboratorium gisras. In fermentasie‐eksperimente het A. tubingensis in kombinasie met S. cerevisiae Y102 gisras die hoogste etanol opbrengs gelewer. Inligting rakende A. tubingensis is beperk relatief tot ander Aspergillii en dit was voorheen aanvaar dat A. tubingensis die hoogste homologie met Aspergillus niger het. Ander verslae – insuitende die huidige studie ‐ het egter gevind dat A. tubingensis nader verwant aan ander Aspergillus spp. in terme van amilolitiese ensieme is. Die α‐amilase geen van A. tubingensis het ‘n homologie van 99.00% met dié van Aspergillus kawachii en die glukoamilase ‘n homologie van 99.26% met dié van Aspergillus shirousami getoon. In die tweede gedeelte van hierdie studie is twee rekombinante S. cerevisae gisrasse gekonstrueer om onderskeidelik die α‐amilase (Atamy) en glukoamilase (Atglu) van A. tubingensis uit te druk. Die kombinasie van die twee rekombinante gisrasse was in staat om die volledige hidrolise en benutting van rou mieliestysel vir die produksie van bio‐etanol deur te voer met ‘n opbrengs van 11.04 g/l wat gelykstaande is aan 98% van die teoretiese opbrengs vanaf stysel met ‘n omskakeling van 52% van die totale rou stysel. Hierdie omskakelingskoers is laer as ander studies wat onderskeidelik 82% en 96% van die teoretiese opbrengs van rou en oplosbare stysel vir α‐ en glukoamilase getoon het. Verder was die kombinasie van die twee gene meer effektief as wanneer slegs een gebruik is, met ‘n 0.32 g/l opbrengs vir Atamy en 2.52g/l vir Atglu. Hierdie het bewys dat die kombinasie van die A. tubingensis meergeskik vir die produksie van bio‐etanol was. Dit het ook bewys dat die beginsel van ‘n amilolitiese gisras wat in staat is om rou stysel te hidroliseer en te fermenteer, inderdaad moontlik is.

L'ARN polymérase III synthétise de nombreux petits ARN non traduits, dont les ARNt et l'ARNr 5S, essentiels à la croissance de toute cellule. Dans ce travail, nous nous sommes intéressés à la régulation de la transcription par l'ARN polymérase III chez la levure Saccharomyces cerevisiae. Nous avons détecté Sub1 sur les gènes de classe III in vivo. Nous avons également observé que Sub1 est capable de stimuler la transcription par l'ARN III reconstituée in vitro avec les facteurs TFIIIB et TFIIIC recombinants et avec l'ARN Pol III purifiée. Sub1 stimule deux étapes de la transcription : l'initiation et la réinitiation facilitée. Des expériences supplémentaires nous montrent que la protéine interagit directement avec TFIIIB et TFIIIC. Enfin, nous avons pu constater que la délétion de Sub1 dans la levure conduit à une diminution de la transcription par l'ARN Pol III en phase exponentielle de croissance. Par la suite, nous avons cherché à déterminer quel lien pouvait exister entre l'activateur Sub1 et le répresseur Maf1 de la transcription par l'ARN Pol III. Enfin, nous avons également souhaité identifier d'autres éléments pouvant interagir avec la protéine Sub1 au cours de sa fonction de régulateur.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is able to sense and respond to changes in its immediate environment. Information regarding the nutritional status of the extracellular environment is sensed by membrane receptor systems and relayed through signalling pathways to the nuclear interior, affecting the transcription of specific genes., Transcription factors, which function downstream of these signal transduction pathways, have to be transported into the nucleus after synthesis in the cytoplasm in order to regulate transcriptional events. Transport into the nucleus occurs in a tightly regulated manner at the nuclear pore complex, which is located in the nuclear membrane, and requires the recognition of transport signal sequences, which are present in the proteins that are to be transported. Signalling pathways control the nuclear accessibility of transcriptional regulators by modifying their respective signal sequences. In response to a limited availability of carbon or nitrogen, cells are able to change their morphology from a unicellular ovoid form to elongated cells attached to each other. This morphological change is associated with daughter cells that remain attached to their respective mother cells following unipolar budding, thus forming filamentous structures referred to as pseudohyphae. The regulation of the development of pseudohyphae is correlated with other physiological processes, such as starch degradation and the invasion of agar-containing media. Mss11p performs a central role in the regulation of the genes required for these processes and it has been shown to specifically regulate the expression of FL011, which encodes a cell surface protein critical for pseudohyphal development, and STA2, which encodes an extracellular glucoamylase functioning in the degradation of starch. The aim of this study was to characterise the functioning of Mss11p. Overexpression analysis indicates that Mss11p functions as an inducer of invasive growth, cell elongation and flocculation. Furthermore, MSS11 deletion improves biomass formation and suppresses the growth defect of yeast from a L:1278b genetic background transformed with the RAS2val19 allele on non-fermentable carbon sources. Biochemical analysis shows that Mss11p is a nuclear protein of approximately 97 kDa in apparent size that is maintained at relatively low levels in yeast. Finally, the data suggest a model in which Mss11p functions as a mediator of the transcriptional regulation of various genes.
AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae is in staat om veranderinge in sy onmiddelike omgewing waar te neem en daarop te reageer. Inligting betreffende die beskikbaarheid van voedingstowwe in die omgewing word vanaf membraan reseptorsisteme deur middel van seintransduksiekaskades na die nukleus herlei, waar die transkripsie van spesifieke gene beïnvloed word. Transkripsie faktore wat stroom af van hierdie seintransduksie funksioneer, moet na die nukleus vervoer word na vervaardiging in die sitoplasma, om sodoende transkripsionele gebeurtenisse te reguleer. Die vervoer van faktore na die binnekant van die nukleus vind onder streng regulering plaas by die nukleêre porie kompleks, wat in die nukleêre membraan gesitueer is. Vervoer vind plaas deur middel van die herkenning van nukleêre lokaliseringsekwense wat in die proteïene wat vervoer word, teenwoordig is. Seintransduksiekaskades beheer die beskikbaarheid van proteïene tot die nukleus deur hulonderskeidelike nukleêre lokaliseringsekwense te modifiseer. Selle is in staat om hul morfologie te verander van 'n eensellige eliptiese vorm tot verlengde selle wat aan mekaar geheg bly in reaksie op die beperkende beskikbaarheid van koolstof of stikstof bronne. Hierdie morfologiese verandering word geassosieer met dogterselle wat ná monopolêre botselvorming aan hul moederselle geheg bly, en dus filamentagtige strukture vorm wat pseudohifes genoem word. Die regulering van die ontwikkeling van pseudohifes word gekorreleer met ander fisiologiese prosesse, soos styselafbraak en die penetrerende groei van selle op agar-bevattende media. Mss11p vervul 'n sentrale rol in die regulering van gene wat vir hierdie prosesse benodig word en reguleer die uitdrukking van FL011, wat kodeer vir 'n selwandproteïen wat krities is vir die ontwikkeling van pseudohifes, en STA2, wat kodeer vir 'n ekstrasellulêre glukoamilase wat vir die afbraak van stysel benodig word. Die doel van hierdie studie was om Mss11p-funksie te karakteriseer. Deur middel van oorproduksie is Mss11p as die induseerder van penetrerende groei, selverlenging en flokkulasie geïdentifiseer. Verder is bevind dat MSS11-delesie lei tot verhoogde biomassa formasie, en dat dieselfde delesie lei tot 'n oorkoming van 'n groeidefek van gis van die 2:1278b genetiese agtergrond wat met die RAS2val19aleel op nie-fermenteerbare koolstofbronne getransformeer is. Biochemiese analise dui daarop dat Mss11p 'n nukluêre proteïen is van ongeveer 97 kDa in oënskynlike grootte, wat teen lae vlakke in gis onderhou word. Die data stel 'n model voor waarin Mss11p as bemiddelaar optree vir die transkripsionele regulering van verskeie gene.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
As amilases são enzimas pertencentes à classe das hidrolases, atuando na estrutura do amido com a hidrólise das ligações glicosídicas dos tipos α-1,4 e α-1,6, do interior das cadeias de amilose e amilopectina, respectivamente. Atualmente são responsáveis por cerca de 30% do mercado mundial de enzimas e apresentam uma vasta gama de aplicações industriais. Os fungos capazes de produzir enzimas amilolíticas podem ser cultivados mediante insumos de baixo custo empregados na fermentação em estado sólido (FES), o que possibilita a valorização dos subprodutos da agroindústria, bem como fornece suporte semelhante ao encontrado pelo micro-organismo no ambiente natural. O presente estudo visa produzir amilases por Rhizopus microsporus var. oligosporus em FES utilizando o farelo de trigo como substrato, purificar parcialmente o extrato bruto enzimático obtido, caracterizar bioquimicamente o extrato enzimático parcialmente purificado e posteriormente realizar a sacarificação pela hidrólise do amido presente na quirera de arroz e fermentação alcoólica por Saccharomyces cerevisiae para produção de etanol. A atividade enzimática do extrato bruto foi 39,8 U/mL o que equivale a 358 U/g de substrato. O índice de purificação, após as etapas de cromatografia foi parcial, mas suficiente para que a caracterização bioquímica do extrato produzido fosse realizada. Estes testes demonstraram faixa ótima no pH 4,0 e pH 5,5, indicando as condições ácidas como as melhores para este estudo. A estabilidade do pH foi ampla variando do pH 3,5 ao pH 8,5 com cerca de 40 a 60% de atividade relativa. A temperatura ótima de atividade enzimática determinada como ideal foi de 60 a 65 °C, porém a enzima mostrou-se termoestável até 60 °C. Os testes do efeito de íons na reação enzimática amilolítica demonstraram que os íons Cu+2, Zn+2, Al+2 e Na+2 comportaram-se como inibidores da atividade. O íon Mn+2 destacou-se por potencializar em...
Amylases are enzymes belonging to the class of hydrolases acting on starch structure with the hydrolysis of glycosidic bonds of α-1,4 and α-1,6 types, in the interior of the chains of amylose and amylopectin, respectively. Currently they are responsible for about 30% of the world market enzymes and show a wide range of industrial applications. Fungi capable of producing amylases can grow by low cost inputs in solid-state fermentation (SSF), which enables the recovery of the agricultural industry by-products and provides support similar to that found by the microrganism in the natural environment. This study aims to produce amylase by Rhizopus microsporus var. oligosporus in solid-state fermentation using wheat bran as substrate partially to purify the crude enzyme extract obtained biochemically characterize the partially purified enzyme extract and subsequently to carry out the hydrolysis saccharification of starch present in broken rice and fermentation of it by Saccharomyces cerevisiae for ethanol production. Enzymatic activity of the raw extract was 39.8 U/mL equivalent to 358 U/g substrate. The purification ratio after the chromatography step was partial, but sufficient for the biochemical characterization of the produced extract was taken. These tests showed optimal range of pH 4.0 to pH 5.5 indicating the acidic condition as the best for this study. The pH stability was wide range of pH 3.5 to pH 8.5 with 40 to 60% relative activity. The optimum temperature for enzyme activity determined as optimum was 60 to 65 °C, but the enzyme was thermostable up to 60 °C. Ion effect of the amylolytic enzyme tests showed that the reaction Cu+2, Zn+2, Al+2 and Na+2 ions behaved like activity inhibitors. The Mn+2 ions distinguished for enhancing at about 60% relative enzymatic activity to hydrolysis without addition of ions. The enzymatic hydrolysis of broken rice using as substrate allowed the complete conversion of starch to reducing sugars ...

Orientador: Pedro de Oliva Neto
Banca: Maria das Graças de Almeida Felipe
Banca: Eutímio Gustavo Fernández Núñez
Resumo: As amilases são enzimas pertencentes à classe das hidrolases, atuando na estrutura do amido com a hidrólise das ligações glicosídicas dos tipos α-1,4 e α-1,6, do interior das cadeias de amilose e amilopectina, respectivamente. Atualmente são responsáveis por cerca de 30% do mercado mundial de enzimas e apresentam uma vasta gama de aplicações industriais. Os fungos capazes de produzir enzimas amilolíticas podem ser cultivados mediante insumos de baixo custo empregados na fermentação em estado sólido (FES), o que possibilita a valorização dos subprodutos da agroindústria, bem como fornece suporte semelhante ao encontrado pelo micro-organismo no ambiente natural. O presente estudo visa produzir amilases por Rhizopus microsporus var. oligosporus em FES utilizando o farelo de trigo como substrato, purificar parcialmente o extrato bruto enzimático obtido, caracterizar bioquimicamente o extrato enzimático parcialmente purificado e posteriormente realizar a sacarificação pela hidrólise do amido presente na quirera de arroz e fermentação alcoólica por Saccharomyces cerevisiae para produção de etanol. A atividade enzimática do extrato bruto foi 39,8 U/mL o que equivale a 358 U/g de substrato. O índice de purificação, após as etapas de cromatografia foi parcial, mas suficiente para que a caracterização bioquímica do extrato produzido fosse realizada. Estes testes demonstraram faixa ótima no pH 4,0 e pH 5,5, indicando as condições ácidas como as melhores para este estudo. A estabilidade do pH foi ampla variando do pH 3,5 ao pH 8,5 com cerca de 40 a 60% de atividade relativa. A temperatura ótima de atividade enzimática determinada como ideal foi de 60 a 65 °C, porém a enzima mostrou-se termoestável até 60 °C. Os testes do efeito de íons na reação enzimática amilolítica demonstraram que os íons Cu+2, Zn+2, Al+2 e Na+2 comportaram-se como inibidores da atividade. O íon Mn+2 destacou-se por potencializar em...
Abstract: Amylases are enzymes belonging to the class of hydrolases acting on starch structure with the hydrolysis of glycosidic bonds of α-1,4 and α-1,6 types, in the interior of the chains of amylose and amylopectin, respectively. Currently they are responsible for about 30% of the world market enzymes and show a wide range of industrial applications. Fungi capable of producing amylases can grow by low cost inputs in solid-state fermentation (SSF), which enables the recovery of the agricultural industry by-products and provides support similar to that found by the microrganism in the natural environment. This study aims to produce amylase by Rhizopus microsporus var. oligosporus in solid-state fermentation using wheat bran as substrate partially to purify the crude enzyme extract obtained biochemically characterize the partially purified enzyme extract and subsequently to carry out the hydrolysis saccharification of starch present in broken rice and fermentation of it by Saccharomyces cerevisiae for ethanol production. Enzymatic activity of the raw extract was 39.8 U/mL equivalent to 358 U/g substrate. The purification ratio after the chromatography step was partial, but sufficient for the biochemical characterization of the produced extract was taken. These tests showed optimal range of pH 4.0 to pH 5.5 indicating the acidic condition as the best for this study. The pH stability was wide range of pH 3.5 to pH 8.5 with 40 to 60% relative activity. The optimum temperature for enzyme activity determined as optimum was 60 to 65 °C, but the enzyme was thermostable up to 60 °C. Ion effect of the amylolytic enzyme tests showed that the reaction Cu+2, Zn+2, Al+2 and Na+2 ions behaved like activity inhibitors. The Mn+2 ions distinguished for enhancing at about 60% relative enzymatic activity to hydrolysis without addition of ions. The enzymatic hydrolysis of broken rice using as substrate allowed the complete conversion of starch to reducing sugars ...
Mestre

Resumo: A principal meta deste trabalho foi a de realizar estudos com leveduras industriais brasileiras utilizadas em usinas, na tentativa de viabilizar o emprego da Tecnologia de Fermentação de Mostos com Altos Teores de Açúcares Fermentecíveis, na produção de etanol combustível. Inicialmente, foram realizados experimentos para obtenção de informações relativas as linhagens, na fermentação de sacarose, maltose e glicose, em meios contendo uma base nitrogenada e suplementado com fontes de nitrogênio com diferentes complexidades estruturais. Os estudos mostraram que as linhagens industriais apresentam diferentes perfis de crescimento nos meios suplementados com diferentes fontes de nitrogênio, que variaram de um simples sal de amônio, a um hidrolisado ácido de proteínas (casaminoácidos) e hidrolisado enzimático de proteínas (peptona). Entretanto, a maior capacidade de acúmulo de biomassa pode não ter um reflexo direto na capacidade de utilização de açúcares e a consequente produção de etanol pelas leveduras industriais. Além da natureza estrutural da fonte de nitrogênio e do tipo de açúcar, a presença do oxigênio, em maior quantidade nos cultivos agitados, interferiu sobremaneira no desempenho fermentativo das leveduras industriais, e até no acúmulo de trealose. No geral, foi a suplementação com peptona que propiciou maior produção de biomassa, preservação da viabilidade celular e consumo mais eficiente da fonte de carbono, em cultivos agitados e não agitados. Os estudos com alta densidade celular tiveram como objetivo definir condições experimentais para a condução da Fermentação de Mosto com Altos Teores de Açúcares Fermentescíveis, e que pudessem propiciar ao final do processo a completa utilização da sacarose, associada á preservação da viabilidade... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In this work studies with Brazilian yeast for ethanol production were conducted in order to verify the possibility of utilization of brazilian yeasts for ethanol production in an industrial process known as Very High Gravity Sucrose Fermentation Technology, to produce wine with high ethanol contends. This technology has the advantage to reduce production costs by increasing fermentation yields. Initially, studies were conducted with sucrose, maltose and glucose fermetation in medium containing YNB, supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that yeast strains vary in their response to nitrogen source complex structure, kind of sugar and to oxygen availabity. In general, under peptone supplementation all strains, in shaking and static conditions, showed higher biomass accumulation, efficient sugar utilization and yeast viability was preserved. Sugar utilization by industrial strains not always was directly correlated with higher biomass accumulation. Trehalose accumulation was also influenced by the structural complexity of nitrogen sources, the kind of sugar and the presence of oxygen. Studies with high cell density were conducted to define experimental parameters for Very High Gravity Sucrose Fermentation, in order to induce at the end of process full sugar exhaustion together cell viability preservation. Complete sucrose utilization was detected only in media with 22 and 25% (w/v) sucrose. In the presence of higher sucrose contends (30% (w/v)), total sucrose exhaustion was obtained in a sugar cane based medium, supplemented with 2% (w/v) peptone. The results described in this thesis suggest that industrial yeasts show differing nitrogen demand, and the utilization of Very High Gravity Sucrose Fermentation technology could be carried only after finding the appropriated nutritional and fermentation conditions
Orientador: José Roberto Ernandes
Banca: João Atilio Jorge
Banca: Jonas Contiero
Banca: Sandra Helena da Cruz
Banca: Luis Henrique Souza Guimarães
Doutor

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

This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.

Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries.
AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

Diacetyl (2,3-butanedione) is an important flavour active, oxidative compound that has significant impact on cellular health as well as financial impact in industrial fermentations. The presence of diacetyl in certain fermented beverages, such as beer, results in an unpleasant butterscotch-like flavour and its concentration needs to be reduced by yeast to below the taste threshold prior to filtration and packaging. This results in significant process inefficiency. Furthermore, diacetyl negatively impacts cellular health and has been associated with neurodegenerative diseases and general cell aging amongst others. The reduction of this compound is therefore essential for cellular health.
Several yeast cell enzymatic mechanisms responsible for diacetyl reduction were identified and characterised, including Old Yellow Enzyme (OYE) isoforms and D-Arabinose Dehydrogenase (ARA1). OYE isoforms displayed different micromolar affinities and catalytic turnover rates for diacetyl and catalysed diacetyl reduction in a biphasic manner. ARA1 catalysed diacetyl reduction in a monophasic manner with a millimolar Michaelis constant.
Knowledge gained in these studies was applied in investigations of diacetyl production and reduction in industrial brewing operations and the enzymatic systems further exploited for the development of a novel enzyme based assay to determine diacetyl concentrations in beer samples. Concentrations as low as 0.2 muM were detectable with high repeatability.

Orientador: Crispin Humberto Garcia-Cruz
Banca: Mauricio Boscolo
Banca: Edson José Fragiorge
Resumo: Os biocombustíveis têm despertado interesse crescente como alternativa para reduzir o impacto ambiental negativo do uso de combustíveis à base de petróleo, proporcionando ainda maior segurança energética entre os países. Em particular, o etanol celulósico produzido a partir de materiais renováveis lignocelulósicos, entre eles os resíduos agroindustriais, se apresenta como mais competitivo. Neste contexto, o presente trabalho abrange o potencial do uso de resíduos de mandioca (Manihot esculenta Crantz) obtidos durante o processamento de fécula como matéria-prima para a produção de bioetanol. Foram realizados experimentos de hidrólise nas concentrações de 0,5% até 5% de ácido sulfúrico (v/v). Os resultados demonstraram que H2SO4 a 2% durante 10 minutos de reação a 121ºC atingiram a maior liberação de açúcares redutores presentes no resíduo (134,84 g L-1 ). As condições ideais para a fermentação alcóolica utilizando a levedura Saccharomyces cerevisiae ATCC 26602 foram pH 6,5 e temperatura de 35ºC, frascos mantidos sem agitação e concentração de açúcar redutor inicial de 50g L-1, resultando em 21,23 g L-1 de etanol com uma produtividade de 1,86 g L h -1 e rendimento teórico de 96,5% após 10 horas de fermentação (0,49g/g açúcar fermentescível). Os resultados indicaram que resíduos de mandioca podem servir como um substrato potencial para a produção de etanol, visto que certos parâmetros fermentativos para a otimização do processo foram pesquisados. Assim, este estudo fornece dados práticos sobre as condições mais adequadas para o aproveitamento destes rejeitos industriais visando a geração de energia combustível "limpa", característica invejável e cada vez mais atrativa num mundo onde cresce a preocupação econômica e ambiental
Abstract: Biofuels has increased interest as alternative to reduce the negative environmental impact of petroleum-based fuels use, providing greater energy security among countries. In particular, cellulosic ethanol produced from renewable lignocellulosic materials including agroindustrial wastes presents as most competitive. In this context, this work has focused on the potential use of cassava waste (Manihot esculenta Crantz) obtained from starch processing as raw material for bioethanol production. Hydrolysis experiments were conducted in concentrations from 0.5% to 5% sulfuric acid (v/v). The results showed that 2% H2SO4 for 10 minutes at 121°C reaction reached the highest release of reducing sugars present in the residue (134.84 g L -1 ). The optimal conditions for the alcohol fermentation using Saccharomyces cerevisiae ATCC 26602 strain has pH 6.5, temperature of 35°C, flasks without stirring and initial reducing sugar concentration of 50g L-1, resulting in 21.23 g L-1 ethanol with a productivity of 1.86 g L h -1 and theoretical yield of 96.5% after 10 hours fermentation (0.49 g/g fermentable sugar). These results indicated that cassava residues may serve as potential substrate for ethanol production, since certain fermentation parameters for process optimization were investigated. This study provides practical information on the most suitable conditions for use of these industrial wastes aiming to generate "clean" energy, an enviable and increasingly attractive feature in a world where economic and environmental concerns are growing
Mestre

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O provável fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e sofre uma modificação pós-traducional única e essencial, em que uma lisina específica é convertida em um resíduo de hipusina. Este fator já foi relacionado ao transporte nucleocitoplasmático, à degradação de mRNA e à proliferação celular. Dados recentes restabelecem uma função para eIF5A na tradução e sugerem a sua atuação na etapa de elongação ao invés de início, como originalmente proposto. Uma vez que o envolvimento de eIF5A com o degradação de mRNA ainda não foi elucidado, tornou-se interessante estudar qual a natureza desta relação. O metabolismo de mRNA é um processo complexo que envolve as etapas da tradução (mRNAs nos polissomos), repressão da tradução (mRNAs acumulados em grânulos de estresse) e degradação de mRNA (mRNAs nos P bodies). Os componentes da maquinaria de degradação de mRNA acumulam-se nos P bodies e, neste trabalho, foi verificado que eIF5A não se localiza nestes corpúsculos. Ainda, foi avaliada a formação de P bodies no mutante tif51A-3, na temperatura não permissiva (38ºC), e foi verificada uma inibição na formação de P bodies. Outros mutantes com defeitos na elongação da tradução, como cca1-1 e eft2H699K, também apresentaram defeito na agregação dos P bodies. Foi avaliada a existência de interação genética sintética entre os mutantes tif51A-1 e tif51A-3 e mutantes de fatores envolvidos com a repressão da tradução e/ou degradação de mRNA. Foi revelada uma supressão parcial do fenótipo de termossensibilidade com nocautes dos genes SBP1, DHH1 e PAT1, que codificam fatores ativadores da remoção do capacete e repressores da tradução. Por outro lado, uma interação sintético doente entre os mutantes tif51A-1 e xrn1Δ foi...
The putative translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into a hypusine residue. This factor has been involved in several cellular processes such as nucleocytoplasmic transport, mRNA decay and cell proliferation. Recent data have re-established a function for eIF5A in translation and suggests a role in elongation rather than initiation as originally proposed. Since the involvement of eIF5A with mRNA decay has not been elucidated it has become interesting to study the aspects of this relationship. mRNA metabolism is a complex process that involves the steps of translation (mRNAs on the polysomes), translation repression (mRNAs accumulated in granules) and mRNA degradation (mRNAs in P bodies). The mRNA degradation machinery accumulates in P bodies and in this study we have verified that eIF5A is not localized inside them. P bodies assembly was evaluated in the tif51A-3 mutant at non-permissive temperature and this aggregation was inhibited. Other mutant strains with defects in translation elongation, such as cca1-1 e eft2H699K, also presented defective P body assembly. The existence of synthetic genetic interactions between the eIF5A mutants, tif51A-1 and tif51A-3, and translation repression and/or mRNA decay mutants was evaluated. A partial suppression of the temperature sensitive phenotype of the eIF5A mutants was revealed when SBP1, DHH1 and PAT1 genes were deleted. Since those proteins are decapping activators and translational repressors these interactions reveal a putative function for eIF5A as an inhibitor of translation repression. However, a synthetic sick phenotype between tif51A-1 and xrn1Δ mutants was observed. Finally, stress granules... (Complete abstract click electronic access below)

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription.
AFRIKAANSE OPSOMMING: Die natuur is hoogs veranderlik en alle organismes, insluitende gis, moet by die omgewing kan aanpas om te kan oorleef. Baie eksterne faktore beïnvloed die ontwikkeling van die gissel. Vir die gis om by spesifieke omgewingstoestande aan te pas, moet die gis op 'n molekulêre vlak: (i) al die omgewingsparameters waarneem; (ii) die waargenome omgewingsparameters as seine na die selkern deur middel van seintransduksieweë gelei; en (iii) transkripsie van gene aktiveer of onderdruk en ensiemaktiwiteit reguleer om sodoende die gepaste molekulêre respons te implementeer. Die beskikbaarheid van voedingstowwe in die omgewing is een van die belangrikste omgewingseine wat eensellige organismes moet kan waarneem. Saccharomyces cerevisiae kan spesifieke ontwikkelingsopsies, na gelang van die voedingstowwe wat beskikbaar is, uitoefen. In groeiomstandighede waar daar 'n oorvloed van voedingstowwe is, verdeel S. cerevisiae d.m.v. mitose en vesprei dit deur die omgewing. Sodra die voedingstowwe uitgeput is, word mitose onderdruk. Diploïede S. cerevisiae inisieer meiose, wat aanleiding tot die vorming van vier spore gee. Hierdie spore bevat slegs die helfte van die ouer se chromosome en kan gevolglik met 'n ander spoor paar om weer 'n diploïede gissel te vorm. Die spore is bestand teen strawwe omgewingstoestande en kan vir lang tye oorleef. Wanneer die spoor aan gunstige groeitoestande blootgestel word, ontkiem dit om aan 'n nuwe giskolonie oorsprong te gee. S. cerevisiae het egter 'n derde ontwikkelingsopsie, naamlik pseudohife-differensiëring. Wanneer die beskikbaarheid van voedingstowwe in die omgewing afneem, maar nog nie uitgeput is nie, ondergaan die gis 'n morfologiese verandering. Hierdie verandering word gekenmerk deur selverlenging, nl. botselle wat slegs aan die een punt van die gissel vorm en dogterselle wat aan die moerderselle geheg bly. Dit lei tot die vorming van kettings van selle wat van die giskolonie af weggroei. Voorts kan die selkettings ook die groeisubstraat binnedring. Dit staan as penetrasie-groei bekend en laat die gis toe om na nuwe voedingsbronne te soek. Hierdie studie het op die elemente van seintransduksieweë, wat by penetrasiegroei betrokke is, gefokus. Sekere komponente van die seintransduksieweë is reeds goed gekarakteriseer, terwyl ander komponente nog grootliks onbekend is. In hierdie studie, word 'n rol vir RME1 in die verbetering van styselafbraak en penetrasiegroei geïdentifiseer. Aangesien Rme1 p voorheen as 'n onderdrukker van meiose geïdentifiseer is, is 'n litetaruurstudie oor die inisiasie van meiose saamgestel. Die faktore wat meiose induseer, naamlik 'n gebrek aan voedingstowwe en die sel se ploïedie, word bespreek. Die regulering van die meester inisieerder van meiosie, IME1, asook die proteïene waarmee Ime1p reageer, is ook in die studie ingesluit. Die eksperimentele deel van die studie behels die genetiese analise van Rme1 p tydens penetrasiegroei en styselhidroliese. 'n Epistase-analise tussen Rme1 p en elemente van die MAP-Kinasemodule, asook van die transkripsie faktore Mss11 p, Msn1p/Mss10p, Tec1p, Phd1p en F108p, is onderneem. Rme1p is bekend om aan die promotor van CLN2 te bind en transkripsie te induseer. Daarom is die selsikliene CLN1 en CLN2 in die studie ingesluit. Die studie dui daarop dat Rme1 ponafhanklik van die MAP-Kinasemodule funksioneer en nie Cln1 p en Cln2p benodig om penetrasiegroei te induseer nie. FL011/MUC1 kodeer vir 'n selwandproteïen wat noodsaaklik vir pentrasiegroei is. Soos in die geval van die bogenoemde faktore, benodig Rme1 p FL011 om penetrasiegroei te kan induseer. Ten spyte van mss11-, msn1-, ttos-, tec1- en phd1- delesies, kan ooruitdrukking van Rme1p die transkripsie van FL011 induseer. In die omgekeerde eksperiment kon die bogenoemde faktore FL011-transkripsie ten spyte van 'n rme1 delesie induseer. Die resultate dui daarop dat Rme1 p nie in 'n hiërargiese pad funksioneer nie, maar dat dit waarskynlik 'n meer algemene rol deur transkripsiemodifisering vervul.

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.
In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes. Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains. The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O etanol combustível produzido no Brasil apresenta alguns problemas tecnológicos que afetam a sua produção em larga escala, entre estes destacamos a contaminação do processo por microrganismos indesejáveis, e a fermentação de mosto com baixa concentração de açúcares. Neste trabalho estudou-se aspectos relacionados com leveduras contaminantes do processo industrial, e o desempenho fermentativo de linhagens industriais utilizadas em mostos concentrados e suplementados com fontes estruturalmente complexas de nitrogênio. Foram utilizados dois meios básicos, um constituido da base nitrogenada YNB, e outro a base de caldo de cana, no qual foi adicionado diferentes concentrações de sacarose. Estes meios foram suplementados com fonte de nitrogênio que variaram de simples sal inorgânico como sulfato de amônio, e preparações comerciais de hidrolizados enzimáticos (peptona) e ácidos de proteínas (casaminoácidos). Três linhagens industriais foram inoculadas em mosto a base de caldo de cana contendo sacarose 22%, 30% e 35% (p/v), e suplementados com fontes de nitrogênio. A 30°C, em cultivos agitados e não agitados, verificou-se que a utilização completa de sacarose só ocorreu no meios suplementados com uma fonte de nitrogênio. No geral a suplementação com peptona foi a que propiciou fermentação mais eficiente, quando comparada com sulfato de amônio, maior acúmulo de biomassa e manutenção da viabilidade celular. Os estudos mostraram que nas condições de cultivo e com inoculação de baixa densidade celular, é necessária a suplementação para completa utilização da sacarose. No meio com sacarose 35% (p/v), atingiu-se um nível de 14 % (v/v) de etanol no cultivo não agitado e suplementado, sugerindo ser possível a obtenção de vinhos com maior quantidade de etanol, desde que...
The fuel ethanol industry in Brazil has some technological problems that affect its production on a large scale, among those are contamination by undesirable microorganisms, and wine with low ethanol concentration. We study aspects of microbial contamination of industrial processes, and the fermentation performance of industrial strains of concentrated musts supplemented with structurally complex nitrogen sources. We used two basic media, one consisting of a nitrogen base YNB, and another based on sugar cane juice, which was added increasing concentrations of sucrose. These media were supplemented with nitrogen sources varying from simple inorganic salt such as ammonium sulfate, and commercial preparations of enzymatic hydrolysates (peptone) and acidic protein (casaminoacids). Three industrial strains were inoculated into based sugar cane juice containing 22% sucrose, 30% and 35% (w/v) and supplemented with nitrogen sources. At 30°C, in shaken cultures, it was found that the efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general supplementation with peptone was the one that led to more efficient fermentation compared with ammonium sulfate, with higher biomass accumulation and maintenance of cell viability. Studies have shown that in conditions and with low cell density nitrogen supplementation was required for almost complete sucrose utilization. In sucrose 35% (w/v), it was achieved a level of 14-15% (v/v) ethanol in nitrogen supplemented cultures, suggesting the possibility of producting wines with higher amounts of ethanol in working conditions approaching the limit of yeast alcohol tolerance. The YNB medium was more appropriate to study the demand for nitrogen for different industrial strains. In this medium was also observed that supplementation with... (Complete abstract click electronic access below)

Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The market for high quality lower alcohol wines is growing globally. Several factors are responsible for this trend, with socio-economic and health concerns being considered as being the most relevant. It is therefore no surprise that in the past three decades many systems have been developed to reduce wine ethanol levels, each with its own strengths and weaknesses. However, current systems are not always cost effective and frequently result in unwanted side-effects. Microbiological methods primarily based on redirecting carbon flux in existing, or novel Saccharomyces and non-Saccharomyces yeast strains, might have the potential to eliminate or reduce such shortcomings. However, little base-line information regarding differences in ethanol yields of existing wine yeast strains, and on the impact of fermentation conditions on such yields is currently available. In this study the ethanol yield of 15 wine yeast strains was investigated in synthetic wine must under varied wine fermentative conditions including changes in yeast assimilable nitrogen, sugar concentration, pH and fermenting temperatures to identify strains that produce lower ethanol yields and conditions that would favour such an outcome. Most strains and conditions resulted in very similar ethanol yields, however in some cases interesting differences were observed. Some of the strains showed significant differences between high and low nitrogen containing must. Results from synthetic must were confirmed in grape must (Sauvignon Blanc, Chardonnay, Shiraz and Cabernet Sauvignon), but no consistent response could be observed. Interestingly the Shiraz fermentations always showed a higher ethanol yield for all strains investigated. This may be due to a parameter (or combination thereof) which was not included as an experimental factor in our study. Glycerol yield was also studied in the grape must experiments and was found to be more significantly condition dependent than ethanol yield. Temperature and glycerol seemed to be directly proportional confirming the results of previous studies. While temperature did increase glycerol production, it was concluded that the redirection of carbon towards glycerol was not substantial enough to have measurable effect on the final ethanol concentration. The most notable differences which were observed were very specific to a particular yeast strain and condition pairing, thus no generally applicable treatment to achieve lower ethanol yields could be established.
AFRIKAANSE OPSOMMING: Deesdae is daar ‘n groeiende mark vir lae alkohol wyne van hoë gehalte. Verskeie faktore is verantwoordelik vir hierdie verskynsel, met sosio-ekonomiese en gesondheidskwessies as die hoof rolspelers. Vir hierdie rede is daar gedurende die laaste drie dekades baie stelsels ontwikkel om wyn etanol vlakke te verlaag, elkeen met voor- en nadele. Meeste van die huidige stelsels is nie koste effektief nie en lei gewoonlik tot ongewenste newe effekte. Mikrobiologiese metodes wat gebaseer is op koolstof vloei veranderinge in wyn gisrasse mag die potensiaal bied om hierdie tekortkominge te verminder of te oorbrug. ‘n Alternatief is om nuwe Saccharomyces en nie-Saccharomyces gisrasse te identifiseer wat laer etanol opbrengste lewer. In hierdie studie is die etanol opbrengste van 15 wyn gisrasse ondersoek in ‘n sintetiese mos in verskeie toestande, bv. veranderde stikstof vlakke, suiker vlakke, pH en temperatuur, om die rasse te identifiseer wat laer etanol opbrengste lewer (asook die toestande wat laer etanol opbrengste bevorder). Meeste rasse en toestande het soortgelyke etanol opbrengste getoon, alhoewel daar in sekere gevalle interessante verskille was rakende sekere rasse wat verskillende resultate lewer in mos met verskillende stikstof vlakke. Die resultate van die sintetiese mos eksperimente was bevestig in druiwe mos van vier kultivars (Sauvignon Blanc, Chardonnay, Shiraz en Cabernet Sauvignon), maar geen algemene tendens kon afgelei word nie. Wat interessant was is die feit dat die Shiraz fermentasies altyd hoër etanol opbrengste gelewer het vir al vier gisrasse wat gebruik is vir hierdie eksperimente. Dit mag wees weens ‘n eksperimentele faktor wat nie bestudeer was in die raamwerk van hierdie projek nie. Die opbrengs van gliserol was ook bepaal in die verskeie eksperimente en daar was gevind dat gliserol opbrengs baie meer kondisie-afhanklik is in vergelyking met etanol. Temperatuur en gliserol het ‘n direkte verbandskap met mekaar getoon, wat die bevindinge van vorige studies bevestig. Alhoewel verhogings in temperatuur wel gliserol produksie vermeerder het, was die effek nie genoeg om ‘n meetbare impak op die finale etanol konsentrasie te hê nie. Verskillende giste in verskeie verskillende fermentasie toestande het soortgelyke etanol opbrengste gelewer. Die mees merkbare verskille wat bevind is was spesifiek tot individuele gisras en kondisie kombinasies, maar geen algemene afleiding kon gemaak word rakende behandelings wat etanol opbrengste kan verlaag nie.

Orientador: Sandro Roberto Valentini
Coorientador: Cleslei Fernando Zanelli
Banca: Paulo Sérgio Rodrigues Coelho
Banca: Flávio Henrique da Silva
Banca: Iran Malavazi
Banca: Carla Columbano de Oliveira
Resumo: O provável fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e sofre uma modificação pós-traducional única e essencial, em que uma lisina específica é convertida em um resíduo de hipusina. Este fator já foi relacionado ao transporte nucleocitoplasmático, à degradação de mRNA e à proliferação celular. Dados recentes restabelecem uma função para eIF5A na tradução e sugerem a sua atuação na etapa de elongação ao invés de início, como originalmente proposto. Uma vez que o envolvimento de eIF5A com o degradação de mRNA ainda não foi elucidado, tornou-se interessante estudar qual a natureza desta relação. O metabolismo de mRNA é um processo complexo que envolve as etapas da tradução (mRNAs nos polissomos), repressão da tradução (mRNAs acumulados em grânulos de estresse) e degradação de mRNA (mRNAs nos P bodies). Os componentes da maquinaria de degradação de mRNA acumulam-se nos P bodies e, neste trabalho, foi verificado que eIF5A não se localiza nestes corpúsculos. Ainda, foi avaliada a formação de P bodies no mutante tif51A-3, na temperatura não permissiva (38ºC), e foi verificada uma inibição na formação de P bodies. Outros mutantes com defeitos na elongação da tradução, como cca1-1 e eft2H699K, também apresentaram defeito na agregação dos P bodies. Foi avaliada a existência de interação genética sintética entre os mutantes tif51A-1 e tif51A-3 e mutantes de fatores envolvidos com a repressão da tradução e/ou degradação de mRNA. Foi revelada uma supressão parcial do fenótipo de termossensibilidade com nocautes dos genes SBP1, DHH1 e PAT1, que codificam fatores ativadores da remoção do capacete e repressores da tradução. Por outro lado, uma interação sintético doente entre os mutantes tif51A-1 e xrn1Δ foi... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The putative translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into a hypusine residue. This factor has been involved in several cellular processes such as nucleocytoplasmic transport, mRNA decay and cell proliferation. Recent data have re-established a function for eIF5A in translation and suggests a role in elongation rather than initiation as originally proposed. Since the involvement of eIF5A with mRNA decay has not been elucidated it has become interesting to study the aspects of this relationship. mRNA metabolism is a complex process that involves the steps of translation (mRNAs on the polysomes), translation repression (mRNAs accumulated in granules) and mRNA degradation (mRNAs in P bodies). The mRNA degradation machinery accumulates in P bodies and in this study we have verified that eIF5A is not localized inside them. P bodies assembly was evaluated in the tif51A-3 mutant at non-permissive temperature and this aggregation was inhibited. Other mutant strains with defects in translation elongation, such as cca1-1 e eft2H699K, also presented defective P body assembly. The existence of synthetic genetic interactions between the eIF5A mutants, tif51A-1 and tif51A-3, and translation repression and/or mRNA decay mutants was evaluated. A partial suppression of the temperature sensitive phenotype of the eIF5A mutants was revealed when SBP1, DHH1 and PAT1 genes were deleted. Since those proteins are decapping activators and translational repressors these interactions reveal a putative function for eIF5A as an inhibitor of translation repression. However, a synthetic sick phenotype between tif51A-1 and xrn1Δ mutants was observed. Finally, stress granules... (Complete abstract click electronic access below)
Doutor

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to cysteine. The second part focuses on the regulation of Hap4, the regulatory subunit of the Hap2/3/4/5 transcriptional factor complex. Many components of the 25-subunit Mediator complex negatively regulate HAP4 expression. Srb8 undergoes post-translational modification in response to changes of the carbon source. Gal11 and Med3 positively regulate HAP4 expression.

A leucemia linfóide aguda (LLA) é considerada uma doença grave dos glóbulos brancos, sendo mais comum e mais agressiva em crianças e adolescentes. O tratamento para a LLA tem avançado devido aos estudos para a otimização de drogas já utilizadas em quimioterapias. Entre essas drogas estão as enzimas L- asparaginases (ASPases) que atuam reduzindo a concentração de L-asparagina (Asn) na corrente sanguínea, impedindo a proliferação das células cancerosas, visto que essas não conseguem sintetizar quantidades apropriadas desse aminoácido. No entanto, o medicamento por ser oriundo de um procarioto causa severas reações alérgicas aos usuário, afim de diminuir a imunogenicidade deste quimioterápico, é importante gerar um biofármaco oriundo de um eucarioto. Neste âmbito, obtivemos a Pichia pastoris recombinante responsável pela produção da enzima ASPase intermembranar, oriunda do gene ASP3 de Saccharomyces cerevisiae. Através do planejamento experimental, foi possível ter um aumento de 5 vezes na atividade obtida na condição inicial. O clone Mut+ alcançou sua melhor atividade de 8,6 U/g de célula nas seguintes condições: 20°C, pH inicial 6 e 1,5% de concentração de indutor.
Acute lymphoblastic leukemia (ALL) is considered a serious disease of white blood cells, is more common and more aggressive in children and adolescents. Treatment for ALL has advanced due to studies for drug optimization already used in chemotherapy. Among these drugs are the enzymes L-asparaginases (ASPases) which act by reducing the concentration of L-asparagine (Asn) in the bloodstream, preventing the proliferation of cancer cells, since these can not synthesize appropriate amounts of this amino acid. However, the drug to be derived from a prokaryote causes severe allergic reactions to the user, in order to decrease the immunogenicity of the chemotherapy, it is important to generate a biopharmaceutical derived from a eukaryote. In this context, we obtained the recombinant Pichia pastoris responsible for producing the enzyme ASPase intermembrane, coming from the ASP3 gene of Saccharomyces cerevisiae. Through the experimental design, it was possible to have a 5-fold increase in activity obtained at the initial condition. The Mut + clone achieved their best activity of 8.6 U/g cell under the following conditions: 20 °C, initial pH 6 and 1.5% of inducer concentration.

Orientador: José Roberto Fernandes
Banca: Daniela Alonso Bocchini Martins
Banca: João Atilio Jorge
Resumo: O etanol combustível produzido no Brasil apresenta alguns problemas tecnológicos que afetam a sua produção em larga escala, entre estes destacamos a contaminação do processo por microrganismos indesejáveis, e a fermentação de mosto com baixa concentração de açúcares. Neste trabalho estudou-se aspectos relacionados com leveduras contaminantes do processo industrial, e o desempenho fermentativo de linhagens industriais utilizadas em mostos concentrados e suplementados com fontes estruturalmente complexas de nitrogênio. Foram utilizados dois meios básicos, um constituido da base nitrogenada YNB, e outro a base de caldo de cana, no qual foi adicionado diferentes concentrações de sacarose. Estes meios foram suplementados com fonte de nitrogênio que variaram de simples sal inorgânico como sulfato de amônio, e preparações comerciais de hidrolizados enzimáticos (peptona) e ácidos de proteínas (casaminoácidos). Três linhagens industriais foram inoculadas em mosto a base de caldo de cana contendo sacarose 22%, 30% e 35% (p/v), e suplementados com fontes de nitrogênio. A 30°C, em cultivos agitados e não agitados, verificou-se que a utilização completa de sacarose só ocorreu no meios suplementados com uma fonte de nitrogênio. No geral a suplementação com peptona foi a que propiciou fermentação mais eficiente, quando comparada com sulfato de amônio, maior acúmulo de biomassa e manutenção da viabilidade celular. Os estudos mostraram que nas condições de cultivo e com inoculação de baixa densidade celular, é necessária a suplementação para completa utilização da sacarose. No meio com sacarose 35% (p/v), atingiu-se um nível de 14 % (v/v) de etanol no cultivo não agitado e suplementado, sugerindo ser possível a obtenção de vinhos com maior quantidade de etanol, desde que... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The fuel ethanol industry in Brazil has some technological problems that affect its production on a large scale, among those are contamination by undesirable microorganisms, and wine with low ethanol concentration. We study aspects of microbial contamination of industrial processes, and the fermentation performance of industrial strains of concentrated musts supplemented with structurally complex nitrogen sources. We used two basic media, one consisting of a nitrogen base YNB, and another based on sugar cane juice, which was added increasing concentrations of sucrose. These media were supplemented with nitrogen sources varying from simple inorganic salt such as ammonium sulfate, and commercial preparations of enzymatic hydrolysates (peptone) and acidic protein (casaminoacids). Three industrial strains were inoculated into based sugar cane juice containing 22% sucrose, 30% and 35% (w/v) and supplemented with nitrogen sources. At 30°C, in shaken cultures, it was found that the efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general supplementation with peptone was the one that led to more efficient fermentation compared with ammonium sulfate, with higher biomass accumulation and maintenance of cell viability. Studies have shown that in conditions and with low cell density nitrogen supplementation was required for almost complete sucrose utilization. In sucrose 35% (w/v), it was achieved a level of 14-15% (v/v) ethanol in nitrogen supplemented cultures, suggesting the possibility of producting wines with higher amounts of ethanol in working conditions approaching the limit of yeast alcohol tolerance. The YNB medium was more appropriate to study the demand for nitrogen for different industrial strains. In this medium was also observed that supplementation with... (Complete abstract click electronic access below)
Mestre

Sistemas do tipo cell surface display vêm sendo desenvolvidos para expressão de proteínas heterólogas ancoradas à superfície celular de microrganismos. Várias aplicações foram reportadas destes sistemas, incluindo o emprego como biocatalizador celular, desenvolvimento de vacinas e biosorventes celulares. Neste trabalho foi desenvolvido um sistema que permite ancoragem da proteína glicoamilase de Aspergillus awamori à superfície da parede celular da levedura Saccharomyces cerevisiae. O gene codificador da glicoamilase com sua seqüência sinal foi fusionado ao fragmento do gene codificador da região C-terminal da proteína Flo1p (Flo428), que foi utilizada como âncora (fragmento CG*FC). As células de levedura foram transformadas com o fragmento híbrido CG*FC e os transformantes foram capazes de degradar amido e liberar glicose. A atividade da glicoamilase não foi detectada no meio de cultura, porém está presente no sedimento celular. Estes resultados demonstram que a glicoamilase foi ancorada à parede celular da nova linhagem recombinante de levedura.
Cell surface display systems have being developed for expression of heterologous proteins anchored to the cell surface of microorganisms. Several applications of these systems have been reported, including employment as whole-cell biocatalysts, development of vaccines and cellular biosorvents. In this work it was developed a system that allows the anchoring of the Aspergillus awamori glucoamylase protein to the cell wall surface of the yeast Saccharomyces cerevisiae. The gene encoding glucoamylase with its secretion signal was fused to the gene fragment encoding the C-terminal region of Flo1 protein, used as an anchor (CG*FC fragment). Yeast cells were transformed with hybrid CG*FC fragment and transformants were able to degrade starch and release glucose. Glucoamylase activity was not detected in the culture medium, but only in sedimented cells. These results demonstrate that glucoamylase was anchored to the cell wall of the new yeast recombinant strain.

Orientador: Sandro Roberto Valentini
Coorientador: Cleslei Fernando Zanelli
Banca: Mário Henrique Bengtson
Banca: Erich Birelli Tahara
Banca: Iran Malavazi
Banca: Flávio Henrique da Silva
Resumo: O fator de tradução 5A (eIF5A) é altamente conservado e essencial para a viabilidade celular. eIF5A é a única proteína conhecida que sofre uma modificação pós-traducional que gera um resíduo de hipusina, essencial para sua função. Apesar de eIF5A já ter sido envolvida em diferentes vias celulares, estudos mais recentes reforçam seu papel no processo de síntese proteica, mais especificamente na etapa de elongação da tradução. Resultados do nosso grupo revelaram uma relação funcional entre eIF5A e o Fator de Elongação da Tradução 2 (eEF2). No intuito de melhor compreender o papel de eIF5A com o processo de elongação da tradução, o presente trabalho buscou aprofundar os estudos de interação física entre eIF5A e eEF2. Foi mostrado que eIF5A não interage fisicamente de forma direta com eEF2 e que a interação física destes dois fatores é dependente de complexos ribossomais. Ainda, foi revelado que eIF5A e eEF2 não estão presentes no ribossomo simultaneamente ou não ocupam o mesmo ribossomo por muito tempo. Além disso, como foi mostrado que o homólogo de eIF5A em bactéria, EF-P, interage físicamente com a proteína ribossomal L1, foi investigada a relação funcional entre eIF5A e L1 em levedura. Os resultados obtidos mostraram uma forte interação genética entre estes fatores e uma possível dependência da presença de L1 para a ligação de eIF5A no ribossomo. Por fim, como foi mostrado que eIF5A é necessária para a tradução de sequências de prolinas, as quais causam stalling do ribossomo durante a tradução, e que outros tipos de stalling do ribossomo recrutam e ativam o complexo Ribossome Quality Control (RQC), foi estudada também a interação funcional entre o eIF5A e os fatores do RQC, Ltn1 e Tae2. Apesar de ter sido identificada interação genética entre mutantes de eIF5A e destes fatores... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The translation factor 5A (eIF5A) is highly conserved and essential for cell viability. eIF5A is the only protein known to contain the essential amino acid residue hypusine, generated by a post-translational modification. Although eIF5A has been involved in different cellular pathways, recent studies have strengthened its role in protein synthesis, more specifically in the translation elongation step. Results of our group revealed a functional correlation between eIF5A and the Translation Elongation Factor 2 (eEF2). To further investigate the role of eIF5A in translation elongation, we carried out new experiments to study the physical interaction between eIF5A and eEF2. eIF5A does not interact directly with eEF2 and their physical interaction is dependent on ribosomal complexes. Moreover, eIF5A and eEF2 do not bind simultaneously to the same ribosome or they do not occupy the same ribosome for a long time. Additionally, as it was shown that the homolog of eIF5A in bacteria, EF-P, interacts physically with the Large Ribosome Protein 1 (L1). We evaluated a functional interaction between eIF5A and L1. The results demonstrated a strong genetic interaction between these factors and a possible dependency of L1 in the eIF5A ribosome binding. Finally, as eIF5A was recently involved in translation of polyproline stretches, which cause ribosome stalling, and other types of ribosome stalling recruit and activate the Ribosome Quality Control (RQC) complex, we also investigated the functional interaction between eIF5A and the RQC factors Ltn1 and Tae2. Although it was identified genetic interaction between mutants of eIF5A and mutants of these RQC factors, ribosome stalled at polyproline residues does not recruit the RQC complex. Curiously, it was observed that eIF5A is important for the formation of cellular aggregates... (Complete abstract click electronic access below)
Doutor

Este trabalho tem como finalidade estudar e estabelecer alguns parâmetros no processo fermentativo descontínuo alimentado de uma levedura recombinante Saccharomyces cerevisiae W303-181, visando aumentar a obtenção da enzima glicose-6-fosfato desidrogenase (G6PDH). Foram feitas a padronização e otimização do preparo de inóculo de S. cerevisiae recombinante. Foram três as condições estudadas. Reduziu-se o tempo de preparo do inóculo de 114 h, da primeira condição, para e 64 e 48 h para a segunda e terceira condições, respectivamente. Essas duas últimas condições mostraram-se adequados para dar continuidade com os processos fermentativos. Foi feito um estudo de otimização da concentração de micronutrientes (adenina, histidina, triptofano e uracila) no meio de cultivo usando a metodologia de superfície de resposta. Concluiu-se que, ao empregar o meio de cultivo cujas concentrações dos micronutrientes tenham sido otimizadas, a atividade específica de G6PDH atingiu 7927 U/L, 3,2 vezes maior que para o meio controle. Estudou-se a influência da constante de tempo (K), na síntese de G6PDH em processo descontínuo alimentado com vazão de alimentação exponencialmente crescente e decrescente, utilizando meio de cultivo otimizado e não otimizado. Os valores estudados de K para vazão de alimentação exponencialmente crescente foram 0,2, 0,3, 0,5, 0,7 e 0,8 h-1 e, para a decrescente, foram 0,2, 0,5 e 0,8 h-1. Dentre os cultivos com vazão exponencialmente crescente e com meio de cultivo não otimizado, encontrou-se para K=0,2 h-1 uma atividade enzimática (558 U/L) 4,1 vezes maior que para a levedura selvagem. Dentre os cultivos nas vazões exponencialmente crescente e decrescente, encontrou-se para a vazão crescente e K=0,2 h-1 os maiores níveis de produção de G6PDH (847 U/L). Foram estudados a influência da concentração inicial de glicose e o tempo de alimentação do processo descontínuo alimentado com vazão de alimentação exponencialmente crescente e K=0,2h-1. Verificou-se que a concentração inicial de glicose não favoreceu a formação de biomassa ou a produção de enzimas. Contudo, determina e as máximas velocidades específicas de crescimento celular e de produção de G6PDH, com as maiores velocidades correspondendo às menores concentrações iniciais. Por fim, foi estudada a influência da concentração de leucina na síntese da G6PDH e verificou-se que teores de leucina entre 0-240 mg/L não influenciaram o crescimento celular ou a produção de enzima nos cultivos estudados.
The purpose of this work is to study and to establish some parameters in the fed-batch process of a recombinant strain of Saccharomyces cerevisiae, aiming to increase the production the enzyme glucose-6-phosphate dehydrogenase (G6PDH). The recombinant S. cerevisiae inoculum preparation was standardized and optimized. Tree methods were studied. The inoculum preparation time was reduced to 114 h, from first method, to 64 and 48 h to second and third methods, respectively. These two last methods were adequate in order to proceed with the fermentation process. It was evaluated the best micronutrients (adenine, histidine, tryptophan and uracil) concentration in the cultivation medium to produce G6PDH, using a response surface methodology. We concluded that using cultivation medium witch optimized micronutrients concentration, the G6PDH activity reach 7927 U/L, 3.2 fold higher than to not optimized medium. The influence of time constant (K) on the G6PDH synthesis was studied at fed-batch bioreactor under exponentially increasing and decreasing feeding rates, using optimized medium cultivation and not optimized medium. The values for K at increasing rates were 0.2, 0.3, 0.5, 0.7 and 0.8 h-1, and for decreasing rates were 0.2, 0.5 and 0.8 h-1. Among K values for exponentially increasing rates with not optimized cultivation medium, K=02 h-1 shows higher G6PDH production (558U/L), 4.1 fold higher than wild yeast. Among cultivations proceeded with exponentially increasing and decreasing feeding rates and using a optimized medium, increasing rate and k=0.2 h-1 shows the higher G6PDH production (847 U/L) too. The initial glucose concentration and feeding time was studied at fed-batch bioreactor under exponentially increasing feeding rate with K=0.2 h-1. It was verified that initial glucose concentration do not favored mass or G6PDH production. Although, it determines the maximum G6PDH production and the maximum growth rates, with higher rates at lowest initial glucose concentration. At the end, the influence of leucin at G6PDH production was evaluated. It was verified that concentrations values between 0-240 mg/L did not showed influence at cell growth or G6PDH production, at the studied cultivations.