The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo. / xiv, 120 leaves : ill. ; 28 cm.

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

Hybridization studies based on the prototrophic selection technique, involving the use of auxotrophic mutants of strains of all accepted species of the genus Kluyveromyces, are reported. Two main groups of mutually interfertile taxa were established within the genus. The first group comprises Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces dobzhanskii, Kluyveromyces drosophilarum, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces phaseolosporus, Kluyveromyces vanudenii and Kluyveromyces wikenii. The second group consists of Kluyveromyces dabzhanskii, Kluyveromyces drosophilarum, Kluyveromyces laotis, Kluyveromyces vanudenii and Kluyveromyces wiokerhamii. Hybrids were also detected in crosses involving Kluyveromyces drosophilarum and Kluyveromyces waltii as well as Kluyveromyces marxianus and Kluyveromyces thermotolerans. In terms of the concept of the biological species and in compliance with the requirements of the International Code of Botanical Nomenclature, taxa which hybridize with Kluyveromyces marxianus and form fertile recombinants at frequencies observed in intraspecific crosses, are accepted as varieties of Kluyveromyces marxianus. Hybridization was observed between Kluyveromyces marxianus var. lactis and the presumed imperfect forms of some Kluyveromyces species, namely Candida kefyr, Candida macedoniensis and Torulopsis sphaerica. Recombination was not detected in crosses involving Kluyveromyces marxianus var. marxianus and representatives of other yeast genera, i.e. Pichia, Saccharomyces, Torulaspora and Zygosaccharomyces. Conclusions regarding the relationship between members of the genus Kluyveromyces, reached on the basis of this investigation are compared with those reported by other workers, who based their investigations on phenotypic characteristics as well as on the determinations of mol % G+C and DNA-DNA homology studies.

Pichia pastoris is a methylotrophic yeast gaining acclamation for its capabili ties in heterologous protein expression. In contrast to other host organisms such as bacteria or mammalian cells, P. pastoris offers many advantages over its counterparts. For example, P. pastoris is cost-effective in that it can grow to high cell densities on simple media. The optional use of a constitutive (GAP) or inducible (A OXI) promoter and the ab ility to perfo1m post-translational protein modifications are additional qualities that make for a powerful heterologous expression system. This study focuses on harnessing the benefits described to develop a high-throughput reporter system for the screening of potential super-secreting mutant strains of P. pastoris. Plasmid constructs were engineered with the lacZ reporter gene, which encodes for the β-galactosidase protein, and fused to the S. cerevisiae MATa signal sequence. Expression plasmids were successfully transformed in P. pastoris strain yGS 115 followed by induction. Western blot analyses confirm the expression of β-galactosidase and colorimetric activity assays further validate enzymatic function. A mutant library containing cis- and/or trans-acting mutations was created by treating P. pas loris clones harboring the β-galactosidase expression plasmid with ultraviolet (UV) radiation. A colorimetric plate assay was combined with a replica-plating system that enabled the screening of thousands of potential super-secreting mutant colonies in a high-throughput format. This study sheds light onto our current understanding of secretion in yeast and further contributes to developing P. pastoris into a valuable heterologous protein expression system.

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

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.

We can now manipulate the genetic material of living organism routinely and cheaply. This has inspired a burgeoning field of synthesis based on DNA as a building block. The development of this new synthetic field has mirrored the trajectory of synthetic organic chemistry from small molecular systems to complex macromolecular assemblies. At first, this field of synthetic biology delivered recombinant proteins that enhanced our understanding of the structure-function relationship of biological macromolecules. Now, as the synthetic tools and analysis methods have come of age, synthetic whole-cell and multicellular systems have come within reach. In Chapter 1 we review the significant advances in DNA synthesis and analysis that have brought us to this point. In this work, we first ask what practical applications will benefit most from the unique qualities of synthetic whole-cell system, such as their ability to replicate, sense and respond with molecular specificity. In Chapter 2, we implement a pathogen detection platform based solely on genetically modified yeast. This approach holds the potential to deliver ultra low-cost sensors that can be used and produced at the point-of-care. In Chapter 3, we develop methods to target these yeast-based sensors for the detection of any peptide biomarker of choice. We next look forward to the potential of synthetic multicellular systems. While natural multicellular systems can be directly manipulated, our ability to rationally build multicellular systems from the bottom-up is still in its infancy. There still remain gaps in the available tools to make and analyze such synthetic systems. In Chapter 4, we leverage the explosion of available genomic databases to uncover a highly extensible set of cell-cell signaling modules. In Chapter 5, we implement ratiometric fluorescent tags to track mixed cell populations in multiplex. Together these components will be useful in implementing and analyzing synthetic communication networks that will be key components of advanced synthetic multicellular systems.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses, bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9, 4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control, although it was not possible to determine whether p-coumaroyl-CoA was produced. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast. / AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

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.

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.