Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols during alcoholic fermentation. Some of these esters and higher alcohols are important for the fruity flavors and therefore the final quality of wine and other fermented beverages. Esters are produced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast, ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by the ATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene. Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate, while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fair amount of information is available regarding the ATF 1 gene, limited information is available on the remaining alcohol acetyltransferases. Only two genes that code for esterases have been identified in yeast, namely lAHI and TIPI. It has also been shown that the balance between alcohol acetyltransferases and esterases is important for the net rate of ester accumulation. Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acid metabolic pathway by decarboxylation and reduction. The transamination of the amino acid to the respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain amino acid transferases, which are encoded by the BATI and BAT2 genes, respectively. In recent years, a strong scientific and industrial interest in the metabolism of flavoractive compounds has emerged, but information regarding the roles of specific enzymes and the physiological relevance of their metabolism remains limited. The aim of this project was to investigate the physiological and metabolic consequences of changes in the expression levels of some of the key enzymes involved in aroma compound production. The consequences of these changes on the chemical composition and the fermentation bouquet of wines and distillates were also investigated. The first part of the section on the results in this dissertation reports on the role and relative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism, namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressed in a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeast strain, VIN13. Table wine and base wines for distillation were prepared with these VIN13 transformed strains. The ester concentrations and aroma profiles of the wines and distillates were analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2 increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethyl caproate, while the overexpression of JAHI resulted in a significant decrease in the concentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate, ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease the concentrations of any of the esters. In most cases, there was a correlation between the increase in esters and the decrease in higher alcohols. The data suggest that yeast balances the amount of different esters produced through alcohol acetyltransferases and esterases, and that, in some cases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and the overexpression of two genes, BATl and BAT2, which encode transaminases that contribute to the metabolism of higher alcohols, were investigated. The genes were both disrupted in a S. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wine yeast strain. The effects of these modifications on the general physiology of the corresponding yeast strains and on higher alcohol metabolism were assessed in a range of growth conditions, including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose as sole carbon source and growth in the presence of various concentrations of amino acids. Table wine and base wines for distillation were prepared with the modified industrial strains and the concentrations of the higher alcohols and the aroma profiles of the wine and distillates were analyzed and compared. Batl deletion seemed to be lethal under the conditions that were created, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpression strains, were investigated. These modifications did not appear to significantly affect the general physiology of the strains. The results obtained indicated that the overexpression of BATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesser extent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2 gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid and propionic acid production, and a modest increase in the level of propanol and isovaleric acid. Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamyl acetate concentrations. Sensory analyses indicated that the wines and distillates produced with the strains in which the BATl and BAT2 genes were overexpressed had more fruity characteristics (peach and apricot aromas) than the wines produced by the wild-type strains. This study offers new prospects for the development of wine yeast starter strains with optimized ester and higher alcohol-producing capability that could assist winemakers in their efforts to consistently produce wine to definable specifications and styles and a predetermined flavor profile. / AFRIKAANSE OPSOMMING: Gedurende fermentasie produseer giste 'n wye verskeidenheid vlugtige aromatiese esters en hoër alkohole. Sommige van hierdie esters en hoër alkohole is belangrik vir die vrugtige geure en dra dus by tot die finale kwaliteit van wyn en ander gefermenteerde drankies. Esters word onderskeidelik deur alkoholasetieltranferases en esterases geproduseer en gehidroliseer. In giste word die ester-sintetiserende aktiwiteite deur twee alkoholasetieltransferases verteenwoordig wat deur die ATFI-en ATF2-gene, asook 'n etanolheksanoïeltransferase wat deur die EHTl-geen, gekodeer word. Dit blyk dat ATFlp en ATF2p verantwoordelik is vir die produksie van etielasetaat en isoamielasetaat, terwyl Ehtl p-etielheksanoaat vanaf etanol en heksanoïel-CoA sintetiseer. Alhoewel daar 'n redelike hoeveelheid inligting t.o.v die ATF I-geen beskikbaar is, is daar weinig inligting oor die res van die aloholasetieltransferases. Slegs twee gene wat vir esterases kodeer, is in gis geïdentifiseer, naamlik IAHI en TIPI. Daar is ook bewys dat 'n balans tussen die alkoholasetieltransferases en esterases baie belangrik is vir die netto-tempo van ester-akkumulasie. Hoër alkohole word gesintetiseer vanaf a-keto sure in die vertakte-ketting aminosuur metaboliese pad deur dekarboksilasie en reduksie. Die transaminasie van die aminosuur na die onderkeidelike a-ketosuur word deur vertakte-ketting aminosuur transferases, geleë in die mitochondrion en sitosol, en gekodeer deur BATl- en BAT2-gene, gekataliseer. In die laaste paar jare het daar 'n sterk wetenskaplike, asook industrïele, belangstelling in die metabolisme van aroma-aktiewe komponente te voorskyn gekom, maar inligting in verband met die rol van spesifieke ensieme en die fisiologiese belangrikheid van hul metabolisme is egter beperk. Die doel van hierdie projek was om die fisiologiese en metaboliese gevolge van veranderinge in die ekspressievlakke van sommige sleutelensieme betrokke by aromakomponent-produksie te ondersoek. Die gevolge van hierdie veranderinge op chemiese vlakke, asook hoe die fermentasie-aroma van die wyne en distillate beïnvloed word, is ook bestudeer. Die eerste gedeelte van die resultate rapporteer oor die rol en relatiewe belangrikheid van die Saccharomyces cerevisiae-ensieme betrokke by estermetabolisme, naamlik Atfl p, Atf2p, Ehtlp, Iahlp en Tiplp. Die gene was ooruitgedruk in 'n laboratoriurnras van S. cerevisiae, BY4742, asook in 'n kommersïele wyngisras, VIN13. Tafelwyne en basiswyne vir distillasie is gemaak met die getransformeerde VIN13-rasse. Die esterkonsentrasies en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Die data het gewys dat die ooruitdrukking van ATFI- en ATF2-gene 'n verhoging in etielasetaat, isoamielasetaat, 2-fenieletielasetaat en etielkaproaat veroorsaak het, terwyl ooruitdrukking van !AHI 'n betekenisvolle afname in etielasetaat-, isoamielasetaat-, heksielasetaat- en 2-fenieletielasetaat-konsentrasies veroorsaak het. Die ooruitdrukking van EHTI het 'n duidelike verhoging in etielkaproaat, etielkaprilaat en etielkapraat veroorsaak en die ooruitdrukking van TIPIhet geen van die esterkonsentrasies verander nie. In die meeste gevalle was daar nie 'n korrelasie tussen die toename in esters en afname in hoër alkohole nie. Die data stelook voor dat die gis 'n balans tussen die verskillende esters handhaaf deur middel van die alkoholasetieltrasferases en esterases, en in sommige gevalle blyk dit dat die ensieme dieselfde funksies het en/of mekaar se aktiwiteit beïnvloed. In die tweede gedeelte van die resultate is die oorsake van delesie en ooruitdrukking van twee gene, BAT1 en BAT2, wat kodeer vir transaminases wat tot hoër alkohol metabolisme bydra, bestudeer. Die gene is uitgeslaan in S. cerevisiae BY4742 en ooruitgedruk in BY4742 en in die wyngisras VIN13. Die effekte van hierdie modifikasies op die algemene fisiologie van die verskillende gisrasse en op hoëralkoholmetabolisme is onder 'n verskeidenheid kondisies bestudeer, naamlik aërobies en anaërobiese groeikondisies, in die teenwoordigheid van glukose of raffinose as die enigste koolstofbron, asook in die teenwoordigheid van 'n verskeidenheid konsentrasies aminosure. Tafelwyne en basiswyne vir distillasie is gemaak met die gemodifiseerde industrïele rasse en die konsentrasies van die hoër alkohole en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Bat1-delesie was dodelik onder die kondisies, daarom is slegs die batlts-tes tesame met die BAT1 en BAT2 wat in die rasse ooruitgedruk is, bestudeer. Die modifikasies het nie 'n beduidende effek op die algemene fisiologie van die rasse getoon nie. Die data het wel getoon dat die ooruitdrukking van BAT1 'n verhoging in isoamielalkohol- en isoamielasetaatkonsentrasies, en tot 'n mindere mate isobutielalkohol- en isobottersuur-konsentrasies, veroorsaak het. Die ooruitdrukking van BAT2 het 'n beduidende toename in isobutanol-, isobottersuur- en propioonsuurkonsentrasies en 'n kleinere toename in propanol- en isovaleriaansuur veroorsaak. Die ooruitdrukking van BAT2 het ook gelei tot 'n afname in isoamielalkohol- en isoamielasetaatkonsentrasies. Sensoriese analises het getoon dat die wyne en distillate wat geproduseer is met die rasse waarin die BAT1 en BAT2 gene ooruitgedruk is meer vrugtige eienskappe (perske- en appelkoos-aromas) getoon het as die wyne wat deur die wildetipe rasse geproduseer is. Die studie lewer nuwe vooruitsigte vir die ontwikkeling van wyngiste met geoptimiseerde ester en hoër alkohol produserende eienskappe wat die wynmakers in staat kan stelom wyne te produseer met gedefinieerde spesifikasies en style en 'n voorafbepaalde aromaprofiel.