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.

M.Tech. / Fungi and their mycotoxins are major contaminants of cereals and maize which is the major staple food for Angolan population. The consumption of fungal and mycotoxins contaminated maize can induce health problems as these mycotoxins are known to induce immune suppression and other chronic diseases known as mycotoxicoses. In addition, to these mycotoxins also induce chronic diseases such as cancers of various types (liver, oesophageal, brain). Mycotoxins also immensely affect organs such as fragility and haemorrhage in tissues, liver-necrosis, bile-duct proliferation, caustic effects, intestinal haemorrhage and diarrhoea, adenomatosis, tremors, inco-ordination, mania, coma, photosensitization, sloughing of the extremities, nephrosis, uremia, infertility, prolonged oestrus, huge implications and infections in people with Human immunodeficiency virus (HIV)- AIDS. There is a very little known about the incidence of mycotoxins in Angola, which are associated with Food, Environment and Health. Therefore, the aim of this scientific novel study in history of Angola was to analyse maize samples obtained from major markets in Angola and to establish their quality with regard to mycotoxins and fungi and well as to establish possible links to the prevalent diseases as well as chronic diseases recorded among Angolan populations. To attain these aims, 60 samples of maize were randomly selected and purchased from open rural and nonregulated informal (dusty and food exposed to open air) markets around Luanda, Angola. Luanda, being the capital city of Angola was found to be the best place to sample, because of the presence of huge markets in which food products as well as maize produced in different provinces are delivered and sold to its high population. Thus, sampling in these markets was representative of the whole country and the results obtained give an approximate the status of the quality of maize consumed in different parts of Angola. The result from this study approximates the status of possible health risks that consumers can be exposed due to consumption of these mycotoxins. The samples were analysed in the Food Environmental and Health Research Laboratories at the University of Johannesburg for fungi and mycotoxins contamination using fungal screening methodologies. In terms of mycotoxins extraction, a multi-mycotoxin (solvent-solvent) extraction, solid phase extraction using SAX column for fumonisins (FBs) and immuno-affinity column (VICAM) were used for confirmation of results. Normal thin layer chromatography (TLC), reversed phase thin layer chromatography (RPTLC) (both for FBs confirmation), fluorometer “VICAM” and high performance liquid chromatography (HPLC) were techniques used for mycotoxins detection, identification and quantification. From the results obtained in this study, fungal isolation revealed contamination of the three most important toxigenic species: Aspergillus, Penicillium, Fusarium spp.

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.

Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.

Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).

Diagnostics serve as the frontline defense for the containment and mitigation of infectious diseases. The emerging synthetic biology field established numerous useful applications of engineered biological systems and networks that led to the development of living biosensors. Significant effort has been made to develop G-coupled protein receptor (GPCR)-based yeast biosensors for applications in drug discovery, environmental monitoring, and clinical diagnostics of small molecules and fungal pathogens. Of the living biosensor chassis, yeast-based biosensors offer exceptional advantages over other in vitro diagnostics, such as long-term storage in a reagent-free and dried dormant state, an engineered colorimetric readout for yes/no result interpretation, and high scalability through industrial fermentation. These advantages could be the next innovation as a low-cost, low-tech, and reliable diagnostic alternative in point-of-care and at-home contexts. Chapter 1 provides background information related to synthetic biology, living biosensors, direction evolution, and point-of-care diagnostics. Chapter 2 covers the development of engineered living yeast as a diagnostic tool for viral infections by tailoring the biosensing recognition element to sense any amino acid-based biomarker of choice via directed evolution. Chapter 3 describes the development of living yeast biosensor for the detection of the pathogenic fungus, Aspergillus fumigatus, in clinical supernatants and patient samples. Chapter 4 describes the progression of a living yeast biosensor for the multi-drug resistant pathogenic fungus, Candida auris, and its detection in clinical culture supernatants and samples.

As the name suggests, synthetic biology designs new biology using human power, knowledge, and creativity. Biology is vast, complicated, and all-inclusive, and so is synthetic biology. I believe synthetic biology is the Utopia of biologists, chemists, physicists, material scientists, engineers,and computer scientists. It is a newly emerged and vastly growing field that can impact and improve our lives in many aspects. I dare to say that anything you see that is done by biology can, in the future, be done better by synthetic biology since, on top of having biology as a teacher and as a template, synthetic biology has the benefit of creative and rational design provided by the human brain. In a way, it is the next step in evolution. In this thesis, we worked on some yeast synthetic biology applications. We used engineered yeasts to create bandages to enhance and accelerate the healing of diabetic wounds, make biosensors for pathogenic bacteria and a small molecule metabolite (glucose) important in diabetic patients, and design a community of cells that could contain artificial intelligence. Chapter 1 gives a short introduction and background information regarding diabetes, wound healing, and advanced healing therapies. Chapter 2 is focused on engineering yeasts to secrete wound-healing proteins and in vitro and cell-based studies on the engineered yeasts and secreted recombinant proteins. Chapter 3 presents two wound dressings that contain engineered live yeasts as active ingredients. This chapter includes further in vitro and cell-based studies to assess the functionality of the designed dressings. Chapter 4 focuses on in vivo experiments to study the wound-healing properties of the designed live yeast dressings. Finally, Chapter 5 presents two other projects: one on live yeast biosensors and one on designing modular smart yeast communities that can do computation based on neural network algorithms.

Les protéines associées aux membranes sont d'une importance cruciale pour la cellule. Cependant, en raison d'une plus grande difficulté de manipulation, les données biochimiques les concernant sont très lacunaires, notamment au point de vue de la formation de complexes entre ces protéines.<p><p>L'objectif global de notre travail consiste à combler ces lacunes et à préciser les interactions entre protéines membranaires chez la levure Saccharomyces cerevisiae et plus précisément, entre les transporteurs. Nous avons commencé notre travail par l'étude d'un jeu de données d'interactions à grande échelle entre toutes les perméases détectées par une méthode de double hybride spécialement adaptée aux protéines insolubles (split ubiquitin). Premièrement, la qualité des données a été estimée en étudiant le comportement global des données et des témoins négatifs et positifs. Les données ont ensuite été standardisées et filtrées de façon à ne conserver que les plus significatives. Ces interactions ont ensuite été étudiées en les modélisant dans un réseau d'interactions que nous avons étudié par des techniques issues de la théorie des graphes. Après une évaluation systématique de différentes méthodes de clustering, nous avons notamment recherché au sein du réseau des groupes de protéines densément interconnectées et de fonctions similaires qui correspondraient éventuellement à des complexes protéiques. Les résultats révélés par l'étude du réseau expérimental se sont révélés assez décevants. En effet, même si nous avons pu retrouver certaines interactions déjà décrites, un bon nombre des interactions filtrées semblait n'avoir aucune réalité biologique et nous n'avons pu retrouver que très peu de modules de protéines de fonction semblable hautement inter-connectées. Parmi ceux-ci, il est apparu que les transporteurs d'acides aminés semblaient interagir entre eux.<p><p>L'approche expérimentale n'ayant eu que peu de succès, nous l'avons contournée en utilisant des méthodes de génomique comparative d'inférence d'interactions fonctionnelles. Dans un premier temps, malgré une évaluation rigoureuse, l'étude des profils phylogénétiques (la prédiction d'interactions fonctionnelles en étudiant la corrééélation des profils de présence - absence des gènes dans un ensemble de génomes), n'a produit que des résultats mitigés car les perméases semblent très peu conservées dès lors que l'on considère d'autres organismes que les \ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2005. / The quality of wine is influenced by a variety of factors, most noticeably the quality of the grapes, winemaking practices and the yeast strains used for alcoholic fermentation. Although several yeast strains are present in the must at the beginning of fermentation, strains of S. cerevisiae quickly dominate and survive alcoholic fermentations. This dominance of S. cerevisiae prompted research that led to the development of a multitude of industrial yeast starter cultures. Starter cultures are usually capable of quick and complete fermentations, with minimal production of deleterious substances such as volatile acidity, H2S, SO2 and ethyl carbamate. Yeast strains should be able to survive the stressful environment created during alcoholic fermentation, whilst possibly offering novel oenological benefits such as pectinolytic activity, killer activity and malic acid degradation. The increased production of volatile esters and higher alcohols may also be desirable, as this will allow the production of wines that are more aromatic. In this study, VIN13 was crossed with S. paradoxus strain RO88 and WE14 by using a micomanipulator. VIN13 was chosen for its fast and complete fermentation ability and moderate aroma production potential. Other factors such as the presence of killer activity and low production of volatile sulphur compounds also favoured the selection of VIN13. S. paradoxus strain RO88 was selected for its ability to degrade malic acid and the favourable impact on aroma production during fermentation. Hybrids between these yeasts may have the potential to produce more aromatic wines, with the added bonus of pectinolytic activity and a strong fermentation capacity. The first crossing yielded 5 hybrids between VIN13 and S. paradoxus strain RO88. Two of these hybrids stood out in the sense that they were able to degrade more malic acid than VIN13 and they also possessed killer and pectinolytic activity. Cinsaut wine was made and the 2 hybrids were shown to have higher aroma compound capacity than the parental yeasts. This was also confirmed during sensory evaluation. The second crossing between VIN13 and WE14 yielded 10 hybrids with low H2S production potential and killer activity. WE14 was selected for its ability to produce very aromatic wines and also the slower fermentation capacity. Hybrids between these yeast may have the potential to produce wines with an increased aromatic content and the fermentation rate might be slower, thereby improving the aroma profile of the wine. After microvinification, 5 hybrids were selected on the basis of fermentation rate differing from that of the parental yeasts and favourable oenological traits, such as fast and complete fermentation, high production of glycerol and low production of volatile acidity. Pinotage wine was made and it was shown that some of the hybrids produced more esters and higher alcohols than the parental yeasts. Sensory evaluation also showed the aroma production potential of the hybrids, as some of the hybrids were shown to score higher for banana, cherry and tobacco characteristics.