Thesis (MSc)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape juice by yeast that originate from the grapes and winery equipment. Research has shown that the population composition and dynamics of these yeasts and other microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora, dominate the yeast population found on the surfaces of grapes, although prevailing Saccharomyces cerevisiae strains complete the fermentation process. The yeast S. cerevisiae is an important factor contributing to the quality of wines and, therefore, the improvement of wine yeasts receives considerable attention worldwide. Apart from classical yeast breeding studies, genetic engineering and recombinant DNA techniques are increasingly being used in strain development research programmes. These techniques might enable the wine yeasts to produce heterologous enzymes that degrade polysaccharides, convert malic acid to lactic acid, increase glycerol production, release roam and flavour compounds, secrete antimicrobial peptides, etc. The release of recombinant yeast strains (genetically modified organisms, GMOs) is subject to statutory approval. Therefore, it is important to answer several questions prior to the use of such genetically improved yeast in the commercial production of wine. For example, will recombinant yeast strains be able to multiply and spread in nature, and will this GMO be able to out-compete the natural microflora because of its newly acquired genetic traits. Since existing commercial wine yeasts are used in the abovementioned strain development research, it is essential to determine already at this early stage to what extent these wine yeast strains survive and spread in nature and to what extent they influence the fermentations of the following vintages. This study is divided into two sections. The aim of the first section is to sample a representative number of yeast strains from various vineyards in different climatological areas, mainly in the Western Cape, South Africa. These yeast strains were identified mainly by electrophoretic karyotyping (contour-clamped homogenous electric field electrophoresis; CHEF). The second part of the study summarises the results obtained when Fourier transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine yeast strains. Sets of data, containing the spectra of the mostly used commercial wine yeast strains, were constructed and used as a reference library. The spectra of the isolated yeast strains were then compared to the reference dataset with specific FT-NIR computer software using mathematical calculations. In conclusion, the two methods used in conjunction with one another proved that the commercial wine yeast strains do not easily disperse from the cellar into the vineyard. The commercial wine yeast strains are also more likely to be found near the cellar and the places where the grape skins are dumped. Therefore, should a recombinant yeast strain be used in winemaking, it would not be dispersed into the vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial population in the environment / AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die samestelling en dinamika van die gispopulasie en ander mikro-organismes baie kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik voltooi. Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag. Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens. Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle wynfermentasies van die daaropvolgende jare beïnvloed. Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om 'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte homogene elektriese veld; CHEF) geïdentifiseer. Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR) spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die kommersiële wyngiste te onderskei. As die twee metodes saam gebruik word vir identifikasie, kan die afleiding gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.

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.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize, together with malted barley. In both processes, the enzymes from the barley are responsible for starch conversion and should always be derived entirely from the malted barley. No exogenous enzymes are allowed to be added to any mashing. The enzymes involved in the conversion process to fermentable sugars, are the aand p-amylases, limit dextrinase and p-glucosidase. Maize, on the other hand, contains no enzyme activity, therefore enzymes need to be added when producing whiskey from maize alone. In other whiskey-producing countries where maize is freely available and cheaper than barley, the use of exogenous enzymes are allowed in the mashing process and is crucial for the formation of fermentable sugars from complex carbohydrates. The cost of the enzymes, however, can push the production cost of whiskey to higher levels. Saccharomyces cerevisiae does not have any amylolytic activity, but is an excellent fermenter and produces favourable organoleptic notes, which makes it very suitable for producing potable spirit. Efforts have been made to genetically improve industrial strains, relying on classical genetic techniques followed by the selection of broad traits, such as ethanol tolerance, absence of off-flavours and carbohydrate/starch utilisation. No strain has thus far been selected for total starch degradation during the fermentation of whiskey mash. Over the last decade, considerable progress has been made in the development of genetically improved strains for the distilling, wine, brewing and baking industries. The expression of heterologous genes introduced a new dimension in approaches to the genetic improvement of industrial strains. It would therefore be cost-effective to use a yeast strain that can produce active and sufficient enzymes to ferment raw starch efficiently to alcohol without lowering the quality of the end product. No such strain has been developed to date, but the continuous improvement of starch-utilising strains has made this goal more achievable. Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria that are important for whiskey-specific strains. The selected strains were transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of its rapid fermentation rate. The genes were integrated into the genomes of the yeast strains and were stable after many generations. Assays showed that a significant increase in enzyme activity was induced in the whiskey strains, compared to the untransformed strains. The strains also showed good fermentation ability in whiskey fermentations, although optimum alcohol production was still not achieved. / AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s. gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~- arnitases, limiet dekstrinase en ~-glukosidase. Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme kan egter die produksiekoste van whiskey verhoog. Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol. Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie. Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie, maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel meer bereikbaar. Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.

Dissertation (PhD(Agric))--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Considerable efforts have been made to improve strains of the wine yeast Saccharomyces cerevisiae through the use of genetic engineering. Although the process is well defined, globally there is much resistance towards the use of genetically modified organisms (GMOs), primarily because little is known about their environmental fate and their potential effect on naturally occurring organisms. The public concern is mainly focused on the uncertainty associated with the impact of the deliberate or accidental release of a GMO into the environment. As a consequence, thére is an urgent need to assess the potential risks involved with the use of this new technology. For the eventual global acceptance of any GMO, it is imperative that the consumer must be convinced that it is ultimately safe for human consumption and the environment. In order to achieve this, certain risk assessment procedures must be performed on each and every GMO that is planned to be released into the environment. Although some of the genetically modified (GM) yeasts that have been developed comply with the strict legislation of most countries and have been cleared by regulatory authorities for commercial use, GM yeasts have not, as yet, been used for the commercial production of GM bread, beer or wine. Nevertheless, the use of GM yeasts in the market appears imminent and there is an urgent need to assess and address the perceived health and environmental risks associated with GM foods. The overall objective of this research was to evaluate key environmental issues concerning the use of GM yeasts. The focus was on comparing the behaviour of specific parental and GM yeast strains in model systems in order to determine whether the GM strains may have any selective advantage, which could lead to their spreading. Specifically, it involved monitoring of the growth behaviour of selected GM yeasts within a vineyard microbial community and in fermentations, as well as the interaction of these yeasts with sand and glass surfaces in an aqueous environment. The GM yeasts under investigation were recombinant strains of a well-known, industrial strain of S. cerevisiae VIN13 expressing an a-amylase (designated GMY1); an endo-p-1,4-glucanase and endo-p-xylanase (designated GMY2); and a pectate lyase and polygalacturonase (designated GMY3). The GM yeasts were mist-inoculated onto individually-contained blocks consisting of one-year old grapevines in a secluded glasshouse environment. Specifically, the numbers and dynamics of GM yeast survival, as well as the effect of an introduced GM yeast on the yeast community dynamics and numbers, were investigated. Overall, it was found that the most prevalent wild yeasts isolated from the grapevines were Rhodo torula, Yarrowia lipolytica, Pichia and Candida spp. VIN13 and the GM yeasts did not affect the overall ecological balance of the microflora on the grapevines. Wild strains of S. cerevisiae were seldom isolated from the grapevines. With a few exceptions, the overall detection of GM yeasts was numerically limited. Co-inoculation of (VIN13+GMY1) and (GMY1+GMY2) revealed detection approximately in the same ratio at which they were inoculated, with small differences in the order of GMY2>GMY1 >GMY3. GM yeasts were rarely isolated from bark and soil samples. Spontaneous fermentation of the grapes harvested from the different treated blocks indicated that the GM yeasts survived on the berries, that the natural fermenting ability of VIN13 was conserved in the recombinant strains, and that the GM yeasts did not have any competitive advantage. The soil environment forms an important part of the biosphere and the transport and attenuation of a GM yeast in this matrix will to a large extent affect their ultimate fate in the environment. In soil, microorganisms either occur as suspended cells in pore water or as biofilms on soil surfaces. Although less extensive than a typical soil yeast, Cryptococcus, epifluorescent staining of biofilms confirmed that VIN13 and GMY1 were capable of existing in a biofilm mode on sand granules and glass. Data on effluent numbers detected in flow cells indicated that GMY1 had no advantage due to the genetic modification and had the same reproductive success as VIN13. These strains either had no difference in biofilm density or GMY1 was less dense than VIN13. When co-inoculated, GMY1 had no negative influence on the mobility of Cryptococcus through a sand column, as well as the ability of Cryptococcus to form biofilms. Furthermore, it was found that GMY1 did not incorporate well into a stable biofilm community on glass, but did not disrupt the biofilm community either. This is the first report of the assessment of the fate of GM strains of VIN13 that are suitable for the wine and baking industry. The investigation of the GM yeasts in this study under different scenarios is a good start to an extensive and necessary risk assessment procedure for the possible use of these GM yeasts in the industry. This study could lead to the provision of much-needed scientific and technical information to both industry and regulating bodies. The outcome of this research is also intended to serve as a basis for information sharing with public interest groups. / AFRIKAANSE OPSOMMING: Aansienlike pogings is reeds aangewend om rasse van die wyngis, Saccharomyces cerevisiae, deur middel van genetiese manipulering te verbeter. Alhoewel hierdie proses goed gedefinieerd is, is daar wêreldwyd heelwat teenkanting teen die gebruik van geneties gemanipuleerde organismes (GMO's). Dit is hoofsaaklik te wyte daaraan dat so min bekend is oor hul lot in die omgewing en hul potensiële effek op die organismes wat natuurlik voorkom. Die publiek is veral besorg oor die onsekerheid verbonde aan die bestemde of toevallige vrylating van 'n GMO in die omgewing. Gevolglik is daar 'n dringende behoefte om die potensiële risiko's in die gebruik van hierdie nuwe tegnologie te bepaal. Dit is van uiterste belang dat die verbruiker oortuig sal word van die veiligheid vir menslike gebruik en die omgewing voordat enige GMO uiteindelik wêreldwyd aanvaarbaar sal word. Om dit te kan bereik sal sekere risiko-bepalende prosedures toegepas moet word op ieder en elke GMO wat beplan word om vry gelaat te word in die omgewing. Alhoewel sommige van die geneties gemanipuleerde (GM) giste aan die streng wetgewing van die meeste lande voldoen en deur die owerhede vir kommersiële gebruik goedgekeur is, word GM-giste nog steeds nie vir die produksie van GM brood, bier of wyn gebruik nie. Ten spyte hiervan, blyk die gebruik van GM-giste onafwendbaar te wees en is daar dus 'n dringende behoefte om die voorspelde gesondheids- en omgewingsrisiko's wat met GM voedsel geassosieer word, aan te spreek. Die oorhoofse doel van hierdie navorsing was om belangrike omgewingskwessies aangaande die gebruik van GM-giste te evalueer. Die fokus was op die vergelyking van die gedrag van spesifieke oorspronklike gisrasse en GM-gisrasse in modelsisteme sodat daar bepaal kon word of die GM-gisrasse enige selektiewe voordele het wat moontlik tot hulonbeheerde verspreiding in die natuur sou kon lei. Dit het spesifiek die monitering van die groei van geselekteerde GMgiste binne 'n mikrobiese gemeenskap op wingerd en in fermentasies behels, asook die interaksie van hierdie giste met grond en glas oppervlaktes in 'n wateromgewing. Die GM-giste wat in hierdie studie gebruik is, was rekombinante rasse van 'n bekende industriële ras van S. cerevisiae, VIN13, wat geneties gemodifiseerd was om 'n a-amylase (aangedui as GMG1); 'n endo-p-1,4-glukanase en 'n endo-B-xilanase (aangedui as GMG2); en 'n pektaatliase en 'n poligalaktorinase (aangedui as GMG3) uit te druk. Die GM-giste is op afsonderlike blokke van eenjaaroue wingerdplante binne-in 'n beskutte kweekhuis gesproei-inokuleer. Daar was spesifiek na die selgetalle en dinamika van die oorlewende GM-giste gelet, asook wat die invloed was van die inokulasie van 'n GM gis op die selgetalle van die natuurlike gisgemeenskap. Daar is bevind dat die wildegiste Rhodotorula, Yarrowia Iipolytica, Pichia en Candida spp die gereeldste van die wingerd geïsoleer is. VIN13 en die GM-giste het nie die ekologiese balans van die natuurlike mikrobiese populasie op die wingerd versteur nie. Wilde rasse van S. cerevisiae is selde geïsoleer vanaf die wingerd. In die meeste gevalle is daar bevind dat wanneer GM-giste opgespoor is, hulle in lae selgetalle voorgekom het. Waar giste saam geïnokuleer was, was die opsporing van (VIN 13+GMY1) en (GMY1 +GMY2) ongeveer in dieselfde verhouding as waarin hul geïnokuleer was, terwyl klein verskille in die orde van GMY2>GMY1 >GMY3 opgemerk is. GM-giste is selde vanaf bas- en grond-monsters geïsoleer. Spontane fermentasies van druiwe wat geoes vanaf die verskillende behandelde blokke is, het daarop gedui dat die GM-giste wel op die druiwe oorleef, dat die natuurlike vermoë van VIN13 om te kan fermenteer in die gemodifiseerde gisrasse behoue gebly het en dat die GM-giste geensins deur die genetiese modifikasies bevoordeel was nie. Grond is 'n belangrike deel van die biosfeer en die verspreiding en aanhegting van 'n GM-gis in hierdie matriks sal sy algehele lot in die omgewing tot 'n groot mate beïnvloed. In die grond kom mikroorganismes as gesuspendeerde selle in poriewater of as biofilms op die oppervlaktes van grond voor. Alhoewel biofilmvorming van VIN13 en GMG1 swakker was as in die geval van 'n tipiese grondgis, Cryptococcus, het epifluoresserende kleuring van hierdie S. cerevisiaegiste bevestig dat VIN13 en GMG1 in staat was om as biofilms op sandkorrels en glas te oorleef. Gebaseer op seltellings in vloeiseluitlaat, kon daar afgelei word dat GMG1 geen selektiewe voordeel geniet het as gevolg van die genetiese modifikasie nie en dat die gis net so reproduktief was as VIN13. Hierdie gisrasse het geen verskil in biofilmdigtheid getoon nie of die biofilmvorming van GMG1 was minder dig as die van VIN13. Wanneer GMG1 saam met Cryptococcus geïnokuleer was, het GMG1 geen negatiewe invloed op die beweeglikheid van Cryptococcus deur 'n sandkolom gehad nie en die vermoë van Cryptococcus om biofilms te vorm is ook nie beïnvloed nie. Daar is verder ook bevind dat GMG1 nie goed binne-in 'n gestabiliseerde biofilmgemeenskap op glas geïnkorporeer het nie, maar dat die gis ook nie die biofilmgemeenskap versteur het nie. Hierdie studie verteenwoordig die eerste ondersoek ooit oor die lot, oorlewing en groeigedrag van GM-wyngiste in biologies-afgesonderde wingerd-, fermentasie-, modelgrond- en modelwater-ekosisteme. Die bestudering van hierdie GM-giste onder verskillende omgewingstoestande in afgeslote ekosisteme lê 'n stewige basis vir verdere ondersoeke en die ontwikkeling van omvattende en noodsaaklike risikobepalingsprosedures betreffende die moontlike toekomstige gebruik van GM-giste in die industrie. Hierdie studie baan die weg tot die verkryging van noodsaaklike wetenskaplike en tegniese inligting oor die veiligheidsaspekte rakende GM-wyngiste en dit kan van groot waarde vir die industrie, owerhede en verbruikerspubliek wees.

No abstract available. / Thesis (M.Sc.Eng.)-University of Durban-Westville, 2000.

Communication is a ubiquitous component of life. While complexity and sophistication vary, both unicellular and multicellular organisms constantly interact with their environment. Unicellular organisms, once thought to be asocial, have since been demonstrated to display a multitude of social interactions and hierarchies. For example, quorum sensing enables a bacterial population to modulate gene expression in response to cell-population density, initiating social behavior and the exchange of resources. In eukaryotes, unicellular ascomycete fungi use mating GPCRs to detect secreted peptide pheromones, initiating changes in gene expression required for mating. An overview of communication in unicellular organisms is presented in Chapter 1. In general, these communication systems are characterized by a high degree of fidelity, and as such have been harvested by synthetic biologists to organize communication in synthetic systems. Quorum sensing modules have been employed for pattern formation and to coordinate biosynthesis processes across a community. However, fungal mating remains underutilized as a source of synthetic biology tools. In this dissertation, we leverage fungal mating G-protein-coupled receptors (GPCRs) and their peptide ligands to build responsive yeast systems. We use genome-mining to identify additional fungal peptide-GPCR pairs, which are then characterized in the yeast Saccharomyces cerevisiae. In Chapter 2, we exploit the high specificity and sensitivity of fungal mating GPCRs to design a yeast whole-cell biosensor that produces a visible output in response to detection of peptide biomarkers. In Chapter 3, we genome-mine additional peptide-GPCR pairs and use them as orthogonal signaling channels to build synthetic yeast communities. Finally, in Chapter 4, we use these synthetic yeast communities to provide sense-and-respond capabilities to an Engineered Living Material (ELM).

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.

No description available.

Yeast are exposed to many physical and chemical stresses when used in large-scale industrial fermentations, particularly the initial stages in which yeast are shifted from anaerobic storage to aerated wort. This work investigated the transcriptional and physiological responses of yeast that had been shifted from anaerobic to aerobic growth conditions. Microarray technology was employed to determine the transcriptional changes that occurred in the first hour of a pilot-plant fermentation compared to the 23rd hour. It was found that over 100 genes were up-regulated initially including genes involved in the synthesis of the essential membrane sterol ergosterol and genes for the protection of cells against oxidative stress. It was also determined that cells which accumulate ergosterol precursors in the absence of ergosterol were more sensitive to exogenous oxidative stresses, indicating a role for ergosterol in oxidative stress tolerance. Aeration of anaerobically grown cells did not affect their growth kinetics or viability. However, anaerobically grown cells were hypersensitive to exogenous oxidative stress compared to their aerobic counterparts. Anaerobic cells that underwent a short period of aeration prior to treatment with hydrogen peroxide generated a tolerance to the oxidant, indicating that the period of aeration produced an adaptive-like response. Microarray analysis of the cells during the period of aeration showed that representative genes from the oxidative stress response family were up-regulated rapidly and it was determined that the response was controlled by the Yap1p and Skn7p transcription factors. Deletion of the transcription factor genes indicated that they were responsible for the creation of tolerance to oxidant. Target gene products of the two transcription factors (Gpx2p, Gsh1p and Trx2p) were shown to be induced during the shift to aeration; however, the glutathione redox balance did not seem to be affected as the cells were shifted from highly reduced to oxidising environments. Unexpectedly, it was discovered that genes involved in the synthesis of amino acids were up-regulated during anaerobic growth and stringently downregulated upon aeration of cells. The transcriptional activator of those genes (Gcn4p) was essential for growth in anaerobic media which included amino acid supplementation.

Two yeast/E.coli shuttle vector plasmids were studied in 1994, termed pIBIDB and pBP33. According to this study, each plasmid should contain at least one ADH2UAS (upstream activation sequence in the alcohol dehydrogenase 2 gene) insert. In the present study, the constructed plasmids were analysed and transformed into laboratory strain yeast. The aim of this project was to identify the orientation, quantity and quality of the insert in the selected plasmids. Methods such as restriction analysis, polymerase chained reaction (PCR), sequencing, plate assays and enzyme assays were used to identify and evaluate the novel inserts. The data presented in this thesis suggest the inserted ADH2UAS fragment did enhance the production of maltose permease and maltase when the transformants were cultivated in maltose and ethanol-glycerol medium. The results suggested that transformants containing two inserts of ADH2UAS had a greater influence on the transformants than a single insert. But the inserts within the vectors and in transformed laboratory stain yeast appeared unstable. This could be due to the method used for plasmid construction and the storage condition of the transformants / Master of Science (Hons)