Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During fermentation, nitrogenous compounds serve as nutrients for the yeasts, which enable their growth, functioning and maintenance of the yeasts cells. From a winemaking perspective, a certain amount of nitrogen is required for the yeasts in order to avoid sluggish or stuck fermentation. Moreover, nitrogen metabolism leads to the production of aroma compounds such as higher alcohols, fatty acids and esters which contribute positively to overall sensory characteristics of wine. Nitrogen metabolism (uptake of ammonium and amino acids) have been extensively studied in Saccharomyces cerevisiae. Nonetheless, the fairly great variances observed between strains in terms of preference for certain nitrogen sources and metabolism thereof are not so well understood. Additionally, these mechanisms nitrogen metabolism of non- Saccharomyces yeasts are even vaguer and simply assumed to be globally similar to those of S. cerevisiae. This study aimed to investigate the uptake of nitrogen compounds (ammonium and individual amino acids) by selected non-Saccharomyces yeasts (Lachancea thermotolerans IWBT Y1240, Torulaspora delbrueckii Biodiva TD291, Pichia kluyveri FrootZen, Metschnikowia pulcherrima IWBT Y1123 and Metschnikowia pulcherrima Flavia) to assess the impact of fermentation kinetics and the production of aroma compounds during sequential fermentations with S. cerevisiae under different initial YAN concentrations, with 300 mg/L, 150 mg/L and 75 mg/L, respectively). Fermentations were performed in a synthetic grape juice medium with pure and sequential fermentations. The data showed that the assimilation of nitrogen compounds were species specific. For example, L. thermotolerans preferred alpha amino nitrogen above ammonia, where the opposite hold true for T. delbrueckii. Notable differences could also be identified for the uptake of certain single amino acids. Irrespective of the initial YAN concentrations during sequential fermentations, the yeasts only assimilated about half of the initial YAN. The non-Saccharomyces yeasts did not influence fermentation performance during sequential fermentations. However, a low initial YAN (75 mg/L) had a strong influence on the fermentation kinetics and aroma compound production. The higher uptake (compare to S. cerevisiae) of specific single amino acids by non-Saccharomyces yeasts (especially L. thermotolerans), can be tentatively correlated with certain aroma compounds produced at the end of fermentation. The results also revealed that agitation could impact overall fermentation performance and aroma compound production. This study contributes to an improved understanding of how different initial nitrogen concentrations affect growth, fermentation performances and aroma compound production of wine-related yeasts under fermentative conditions. Moreover, the uptake of single amino acids by selected non-Saccharomyces yeasts had also been identified, which is a good starting point to better understand non- Saccharomyces yeasts nitrogen requirements which may be used for the optimization of nitrogen source addition, during alcoholic fermentation, when used in mixed fermentations in order to ensure a complete alcoholic fermentation. To the best of our knowledge, the uptake of single amino acids and YAN consumption by selected non-Saccharomyces yeasts under fermentation conditions tested, have never been studied before. / AFRIKAANSE OPSOMMING: Tydens wynfermentasies dien talle stikstof komponente as voedingstowwe vir wyngis wat hul groei, funksie en onderhoud bevorder. Van `n wynmaak perspektief word daar `n sekere hoeveelheid stikstof benodig deur die wyngis om te verhoed dat slepende of onvolledige fermentasies plaasvind. Verder lei stikstofmetabolisme na die produksie van aroma verbindings, soos hoër alkohole, vlugtige vetsure en esters wat positief bydra tot die sensoriese karaktereienskappe van wyn. Die stikstofmetabolisme (opneem ammonium en aminosure) is deeglik nagevors in die wyngis Saccharomyces cerevisiae, maar die klein variasies waargeneem tussen die gisras in terme van die voorkeur van sekere stikstof komponente is egter nog onduidelik. Daarbenewens is die stikstofmetabolisme nog meer onbekend in nie- Saccharomyces wyngis en word dit oor die algemeen aanvaar dat die werking van die stikstofmetabolisme dieselfde is as in S. cerevisiae. Hierdie studie het gestreef om die opneem van stikstof komponente (ammonium en aminosure) te ondersoek van uitverkiesde nie-Saccharomyes gis (Lachancea thermotolerans IWBT Y1240, Torulaspora delbrueckii Biodiva TD291, Pichia kluyveri FrootZen, Metschnikowia pulcherrima IWBT Y1123 and Metschnikowia pulcherrima Flavia) deur te bepaal wat die impak is op die groei-kinetika en op die produksie van aroma komponente gedurende gemengde kultuur fermentasies met S. cerevisae onder verskillende aanvangs assimileerbare stikstof (300 mg/L, 150 mg/L en 75 mg/L). Fermentasies is in sintetiese druiwemos uitgevoer vir beide enkel en gemengde kultuur fermentasies. Die resultate demonstreer dat die assimilasie van stikstof ras spesifiek was. Byvoorbeeld, L. thermotolerans verkies alfa amino stikstof bo ammonium waar die teenoorgestelde waar is vir T. delbrueckii. Beduidende verskille is ook waargeneem vir die opneem van sekere individuele aminosure. Die wyngis het steeds net die helfte van die assimileerbare stikstof opgeneem gedurende gemengde kultuur fermentasies ongeag die aanvangsstikstof konsentrasies. Die nie-Saccharomyces gis het nie die fermentasie kinetika beïnvloed tydens gemengde kultuur fermentasies nie. Daar was egter ook waargeneem dat `n lae assimileerbare stikstof (75 mg/L) `n negatiewe invloed op die fermentasie kinetika sowel as aroma produksie gehad het. Die hoër opname (vergelyking met S. cerevisiae) van sekere aminosure deur nie-Saccharomyces gis, kan tydelik gekoppel word aan die produksie van spesifieke aroma verbindings aan die einde van fermentasies. Die resultate het ook gewys dat die toepassing van skud `n impak het op die fermentasie kinetika sowel as die produksie van aroma komponente. Die studie dra by om beter te verstaan van hoe verskillende aanvangsstikstof die groei, fermentasie kinetika en aroma produksie beïnvloed onder fermentasie kondisies. Die opneem van sekere aminosure deur nie-Saccharomyces gis word ook beskryf, wat `n goeie beginpunt is om beter te vertaan wat die stikstof vereistes vir die geselekteerde wyngis is, wat gebruik kan word vir die optimisering van stikstofaanvullings, sodat die risiko van probleemfermentasies verlaag sal word. So ver as wat ons kennis strek is die opneem van aminosure en die gebruik van assimileerbare stikstof deur nie-Saccharomyces wyngis onder fermentasie kondisies nog nie ondersoek nie.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a previous study were identified to strain level and found to belong to the yeast species Candida pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in red wine, and against several strains of the genus Brettanomyces on white and red grape juice medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete killer toxins, which can play a role in yeast microbial interactions under winemaking conditions. The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5 and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during winemaking did not affect the activity and stability of these killer toxins. Although, the killer toxins differed with regards to their biochemical and environmental stability and activity, they were found to have a similar mode of action. The killer toxins induced a fungistatic effect on B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by K. wickerhamii. According to the author’s knowledge this is the first report on the identification of novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed provides great research scope in this field. The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase) and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were identified as the potential toxins, but their killer activity could not be confirmed. These findings suggest that hydrolytic enzymes possess killer activity, as previously reported in literature. However, further investigation is needed to identify the killer toxins characterized in this study. / AFRIKAANSE OPSOMMING: In die huidige studie is twee isolate wat in ’n vorige studie “killer” aktiwiteit teenoor verskeie wyngisspesies vertoon het, tot op rasvlak geïdentifiseer en daar is gevind dat hulle aan die gisspesie Candida pyralidae behoort. Die geïdentifiseerde gisrasse en ’n Kluyveromyces wickerhamii gisras wat as kontrole gebruik is, het “killer” aktiwiteit getoon teenoor B. bruxellensis, wat bekend is vir sy bederfkarakter in rooi wyn, en ook teenoor verskeie rasse van die genus Brettanomyces in wit en rooi druiwesapmedium. Die “killer” giste het nie die groei van óf S. cerevisiae óf van die melksuurbakteria Oenococcus oeni en Lactobacillus plantarum-rasse geïnhibeer nie. Giste word berig om “killer” gifstowwe uit te skei, wat ’n rol kan speel in gis mikrobiese interaksies onder wynbereidingstoestande. Die C. pyralidae-rasse is gevind om twee nuwe “killer” gifstowwe af te skei, wat CpKT1 en CpKT2 genoem is. Hierdie “killer” gifstowwe was stabiel en aktief onder wynbereidingstoestande, pH 3.5 - 4.5 en temperatuur tussen 15 en 25°C. Die etanol- en suikerkonsentrasies wat onder wynbereiding voorkom, het nie die aktiwiteit en stabiliteit van hierdie “killer” gifstowwe beïnvloed nie. Hoewel die “killer” gifstowwe met betrekking tot hulle biochemiese en omgewingstabiliteit en aktiwiteit verskil het, is daar gevind dat hulle ’n eenderse modus van aksie het. Die “killer” gifstowwe het ’n fungistatiese effek op B. bruxellensis sensitiewe selle geïnduseer, buiten dat dit aan die selwand van die sensitiewe selle gebind het, en het seloppervlak- en plasma-membraanskade geïnduseer, net soos die Kwkt “killer” gifstof wat deur K. wickerhamii afgeskei is. So ver die skrywer weet, is hierdie die eerste verslag van die identifisering van nuwe “killer” gifstowwe wat deur C. pyralidae rasse afgeskei word wat uit ’n wynomgewing geïsoleer is, asook van die identifikasie van die modus van aksie van “killer” gifstof op B. bruxellensis selle. Dit verbreed dus beslis die navorsingsomvang van hierdie gebied. Die eksoproteome, bestaande uit die “killer” gifstowwe Kwkt, CpKT1 en CpKT2, het die teenwoordigheid van ekso-glukanases en glukosidases onderskeidelik onthul. Die ensieme KwExg1 (eksoglukanase) en KwSun4 (glukosidase) wat vanuit K. wickerhamii se eksoproteoom herwin is, is as die potensiële gifstowwe geïdentifiseer, maar hulle “killer” aktiwiteit kon nie bevestig word nie. Hierdie bevindings suggereer dat hidrolitiese ensieme “killer” aktiwiteit besit, soos voorheen in die literatuur berig is. Verdere ondersoeke word egter benodig om die “killer” gifstowwe wat in hierdie studie gekarakteriseer is, te identifiseer.

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours. / AFRIKAANSE OPSOMMING: L-Appelsuur en wynsteensuur is die mees prominente organiese sure in wyn en speel 'n kritiese rol in die wynbereidingsproses en organoleptiese wynkwaliteit, insluitende die fisiese, biochemiese en mikrobiese stabiliteit van wyn. Die produksie van hoë-kwaliteit wyne berus op die vermoë van 'n wynmaker om die suurinhoud korrek aan te pas om sodoende 'n gebalanseerde produk met optimale geur en kleur te produseer. Saccharomyces rasse kan gewoonlik nie appelsuur volledig tydens alkoholiese gisting benut nie en dra dus nie noemenswaardig tot 'n verlaging van die totale suurinhoud van wyn by nie. Die mate van appelsuur afbraak deur Saccharomyces wissel egter van ras tot ras. Sekere Saccharomyces rasse kan 'n groter persentasie appelsuur afbreek, maar die onderliggende rede vir hierdie verskynsel is onbekend. Die onderliggende meganismes vir hierdie verskynsel is gedurende hierdie studie uitgelig na afloop van voorlopige transkripsionele regulerings studies op die malaatensiemgeen. In teenstelling hiermee kan S. pombe tot 29 gIl appelsuur via die malo-alkoholiese padweg afbreek waartydens appelsuur na pirodruiwesuur en CO2, en uiteindelik na alkoholonder fermentatiewe toestande omgeskakel word. Verskeie redes vir die swak afbraak van appelsuur deur Saccharomyces cerevisiae is voorgestel. Eerstens beskik S. cerevisiae nie oor 'n meganisme vir die aktiewe transport van appelsuur, soos in die geval van S. pombe nie, en is aangewese op die stadige opname van appelsuur deur eenvoudige diffusie. Tweedens het die S. cerevisiae malaatensiem 'n baie laer substraataffiniteit vir appelsuur (Km = 50 mM) in vergelyking met die van S. pombe (Km = 3.2 mM), wat verder bydra tot die swak afbraak van appelsuur in S. cerevisiae. Laastens dra die mitochondriale ligging van die S. cerevisiae malaatensiem in teenstelling met die sitoplasmiese ligging van die S. pombe malaatensiem, verder by tot die swak afbraak van appelsuur, aangesien die mitochondria onder fermentatiewe toestande negatief gereguleer word. Die malaatpermease geen (maely en malaatensiem geen (mae2) van S. pombe is gevolglik gekloneer en heteroloog in 'n laboratoriumras van S. cerevisiae onder die beheer van die konstitutiewe 3-fosfogliseraat kinase (PGKI) promoter- en termineerdervolgordes uitgedruk. 'n Sterk malo-alkoholiese fenotipe was duidelik tydens fermentasies met die rekombinante gis in sintetiese en Chardonnay druiwemos, met 'n gepaardgaande verhoging in alkoholvlakke. Funksionele uitdrukking van die malo-alkoholiese gene van S. pombe in 'n S. cerevisiae laboratoriumras het die weg vir die genetiese modifisering van industriële wynrasse van S. cerevisiae vir kommersiële wynfermentasie gebaan. Om 'n integrale deel van die gis te word, moet die malo-alkoholiese gene stabiel in die genoom van industriële wynrasse geïntegreer word. Genetiese manipulering van industriële wynrasse word egter bemoeilik deur die homotalliese, multi-ploïediese en prototrofiese aard van industriële Saccharomyces rasse. Transformasie en integrasie van heteroloë gene in industriële Saccharomyces rasse vereis die gebruik van dominante merkers, bv. weerstandbiedendheid teen antibiotika of ander gifstowwe. Integrasie van hierdie merkers in die gisgenoom is egter nie vir kommersiële toepassing aanvaarbaar nie weens die afwesigheid van langtermyn risikobepalings en verbruikersweerstand. Tydens hierdie studie is daar dus gepoog om industriële wynrasse met 'n unieke strategie geneties te verbeter sodat slegs gis-DNA tydens die integrasie van die S. pombe mae1 en mae2 uitdrukkingskassette in die gisgenoom opgeneem word. Die Malo-alkoholiese integrasiekasset wat slegs die S. pombe mae1, mae2 oopleesrame en die S. cerevisiae PGK1 promoter en termineerdervolgordes bevat, is in die URA3 lokus van Saccharomyces bayanus ECll18 geïntegreer tydens parallelle transformasie met 'n 'phleomycin' weerstandbiedendheidsplasmied. Na seleksie van transformante op 'phleomycin' -bevattende media, is die S. bayanus EC 1118 transformante in nieselektiewe kondisies opgegroei sodat verlies van die 'phleomycin' plasmied kon plaasvind. Integrasie van die mae1 en mae2 uitdrukkingskassette is bevestig en kleinskaalse fermentasies in sintetiese en druiwemos het 'n vinnige en doeltreffende afbraak van appelsuur in die vroeë fases van die alkoholiese fermentasie getoon. Integrasie en uitdrukking van die malo-alkoholiese gene in S. bayanus ECl118 het geen nadelige effek op die fermentasievermoë van die gis getoon nie, terwyl sensoriese en chemiese ontleding van die Chardonnay wyne 'n verbetering in aroma relatief tot die kontrole wyne getoon het, met die afwesigheid van enige afgeure.

Orientador: Marco Antonio Tecchio / Banca: Maria Fernandes Moura / Banca: Regina Marta Evangelista / Banca: Evandro Henrique Schinor / Banca: Igor Otávio Minatel / Resumo: Dependendo do sistema de condução e do porta-enxerto empregado no cultivo da videira, as características de produção e parâmetros qualitativos dos frutos podem ser alterados. Dessa forma, objetivou-se com este estudo, avaliar as características de produtividade, produção, qualidade físico-química dos cachos e bagas, e bioquímica das cascas das cultivares de uva Syrah, Sauvignon Blanc e Merlot enxertadas nos porta-enxertos 'IAC 766' e '106-8 Mgt' nos sistemas de condução de espaldeira baixa e alta sendo o experimento conduzido no Centro de Frutas do IAC. O delineamento experimental utilizado para cada cultivar copa foi em blocos casualizados, em esquema fatorial 2x2, com 4 repetições. Os tratamentos foram representados por dois sistemas de condução (espaldeira baixa e espaldeira alta) e dois porta-enxertos ('IAC 766' e '106-8 Mgt'). Os sistemas de condução em espaldeira baixa ou alta, bem como os porta-enxertos 'IAC 766' e '106-8 Mgt' não influenciaram na produção, produtividade e na qualidade física dos cachos da cv. Syrah. Obtiveram-se poucas variações na qualidade do mosto das uvas da cv. Syrah, entretanto, o sistema de condução em espaldeira baixa combinado com o uso do porta-enxerto 'IAC 766' proporciona maior porcentagem de açúcares redutores. Utilizando-se o porta-enxerto '106-8 Mgt' no sistema de condução espaldeira baixa apresentou maior teor de antocianinas. Foi observado para a cultivar Syrah, sendo possível a recomendação de ambos os porta-enxertos e sistemas de co... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Depending on the conduction system and the rootstock used in vine cultivation, the production characteristics and qualitative parameters of the fruits may be altered. Thus, the objective of this study was to evaluate the characteristics of productivity, production, physicochemical quality of the clusters and berries, and biochemistry of the bark of the grape cultivars Syrah, Sauvignon Blanc and Merlot grafted on the rootstocks ' IAC 766 ' and ' 106-8 Mgt ' in the low and high spree conduction systems being the experiment conducted at the IAC Fruit Center. The experimental design used for each cultivar canopy was in randomized blocks, in a 2x2 factorial scheme, with 4 replications. The treatments were represented by two conduction systems (low spread and high spread) and two rootstocks (' IAC 766 ' and ' 106-8 Mgt '). The conduction systems in low or high spreads, as well as the rootstocks ' IAC 766 ' and ' 106-8 Mgt ' did not influence the production, productivity and physical quality of the clusters of CV. Syrah. There were few variations in the quality of the grape must of the CV. Syrah, however, the low-spreaded conduction system combined with the use of the rootstock ' IAC 766 ' provides a higher percentage of reducing sugars. Using the rootstock ' 106-8 Mgt ' in the low-spreading conduction system presented higher anthocyanin content. It was observed for the cultivar Syrah, being possible the recommendation of both rootstocks and conduction systems. The CV. Sauvignon Blanc grafted on the rootstock ' 106-8 Mgt ' showed higher production, productivity, number of clusters and berries and greater fresh mass of clusters and antioxidant capacity (DPPH) in the high spreading system, and higher content of polyphenols and Flavonoids in low spreis. The rootstock ' IAC 766 ' provided higher content of reducing sugar and antioxidant capacity (FRAP) and the high scatting system higher content of flavonoids ... / Doutor

Bibliography: p. 331-340. / x, 340 p. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Using sensory ranking, studies aroma variability of a chardonnay and a cabernet sauvignon matured in new oak barrels. Oak wood-derived volatile compounds are analysed using gas chromatography, to suggest which natural and cultural variables are involved in each aroma variation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1998

Includes bibliographies. This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes.

In an effort to understand the basis for sulfite detoxification in S. cerevisiae, the functions of two genes were analyzed. SSU1, which encodes a plasma membrane protein, was found to be required for efficient sulfite efflux. FZFl-4, a dominant allele of a transcriptional activator of SSUl, was also found to be involved in efficient sulfite efflux. Analysis of an SSUl promoter-lacZ fusion showed that FZFl-4 conferred sulfite resistance through hyperactivation of SSUl. Efflux assays in cells expressing multicopy SSUl or FZFl-4 suggested that Ssulp specifically mediates efflux of the free form of sulfite. Sulfite resistance, mediated by either FZFl-4 or multicopy SSUl, was found to be a useful marker for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZFl-4 was found to be more efficient than multicopy SSUl, and in the case of the laboratory strains, was found to be about half as efficient a selectable marker as URA3. Sulfite transport was studied to clarify the mechanism of sulfite uptake in S. cerevisiae. The kinetics of uptake were saturable, indicating a carrier-mediated process. Uptake was significantly reduced in cells pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or 2,4-dinitrophenol (DNP), both of which dissipate proton gradients. Extracellular alkalization was observed during sulfite uptake. These findings suggest that an anionic form of sulfite, HSO₃, is taken up by carrier-mediated proton symport. As an alternative to costly disposal of spent cherry brine, a sulfite-containing waste stream generated during maraschino cherry processing, brine was tested as a substrate for ethanol production by S. cerevisiae. Initially, the toxic level of sulfite in brine was reduced by raising brine pH to 8.5 with Ca(OH)₂ to precipitate calcium sulfite. Because the alkalization was found to result in a 10-fold reduction of phosphorus, brine was subsequently titrated with phosphoric acid to pH 6.0 prior to inoculation with S. cerevisiae. All strains of S. cerevisiae tested were able to efficiently ferment all lots of Ca(OH)₂-treated and phosphorus-enriched brine. / Graduation date: 2000

Thesis (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on February 3, 2010. Major professor: W. Keith Patterson, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture." "November 2009." Includes bibliographical references (p. 68-70).

Thesis (doctoral)--Universität Hohenheim, 2002. / Includes bibliographical references (p. 109-114).