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.

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.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries. / AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

The aims of the project were twofold. The initial objective of the study, based on previous results, was to develop an economically viable methodology for immobilizing yeast cells for the treatment of heavy metal-laden waste water. The non-viable yeast cross-linked by 13% (w/v) formaldehyde/1N HNO₃ exhibited satisfactory mechanical strength and rigidity in a continuous-flow column operation. No apparent disruption of the biomass after repeated use was observed. The cost of immobilizing 1kg dry yeast pellets was estimated at less than US$I. Zn uptake capacity of FA-cross-linked pellets, on batch trials, remained similar to that of raw yeast, reflecting that the immobilizing procedure did not hinder its metal removing capacity. In column studies, cation metals were effectively removed by the yeast pellets from aqueous solution at natural pHs, and then recovered completely by washing the pellets in situ with O.1M HCl. The recovered metals were concentrated in such small volumes that recycling or precipitation of them was facilitated. The metal uptake capacity of the regenerated biomass remained constant in comparison with cycle 1, indicating that reuse of the yeast would be possible. In the case of Cr⁶⁺, a gradual breakthrough curve of Cr in the column profile was noted, with a simultaneous reduction of Cr⁶⁺ to Cr³⁺. However, Cr⁶⁺ in the effluent can be markedly minimised either by accumulation onto the biomass or reduction to its trivalent form. Desorption of bound Cr⁶⁺ with either alkali or salt could not accomplish the regeneration of the biomass. A combination of reduction and desorption with FA/HNO₃ appeared promising in regeneration of the saturated biomass at 4°C. The metal sorption capacities of the yeast pellets, on a batch or a fixed-bed system are relatively lower than that of documented sorbents. Apparently more of the yeast pellets would be required for treating a certain volume of waste effluent, than with other sorbents. Therefore Azolla filiculoides was examined as a suitable sorbent for this purpose. This constitutes the second part of the project. Azolla filiculoides, a naturally-abundant water fern, was screened for its metal sorption and recovering capacities, mechanical stability, flow-permeability and reusability. The azolla biomass appeared to have fulfilled the required mechanical criteria during the repeated sorption-desorption column operations. It is water-insoluble and appears flexible under pressure when rinsed with water. These characters are of crucial importance in a continuous-flow system since a column can be operated at high flow rates without apparent compact of the biomass and pressure loss. Therefore, immobilization of the biomass can be avoided. The sorption isotherm data, obtained from batch removal of Cr⁶⁺, showed that the sorption process was effective, endothermic and highly pH dependent. Considerable amounts of Cr⁶⁺ were accumulated at the optimum pHs of 2-2.5. Column sorption of Cr⁶⁺ at a low flow rate and pH of 2.5 showed optimum performance with a total Cr uptake of 50.4mg/g at 60% saturation of the biomass. Removal of Cr⁶⁺ from an electroplating effluent using an azolla column was deemed reasonably satisfactory, although the uptake declined slightly. Desorption of bound Cr⁶⁺ with various desorbents was incomplete, which resulted in a low regeneration efficiency of about 50%. However, removal and recovery of Cr³⁺ using the azolla column was than that of Cr⁶⁺. Desorption of Cr³⁺ from the spent biomass column was accomplished with the recovery of 80% using O.5N H₂SO₄, The regeneration efficiencies for Cr³⁺ removal were up to 90% and demonstrated that the biomass is reusable. Cation metal uptake capacities of azolla, obtained either from batch or column experiments, are reasonably high in comparison with other sorbents. The uptake of Ni or Zn ions from solution is pH dependent showing the optimum pH of around 6 to 6.5, under the current experimental conditions. The sorption kinetics for cation metals was rapid with about 80% of the bound Ni ions being taken up in the first 10 min. The character of rapid binding is extremely important in a column sorption process, especially on a large scale since it favours an optimum uptake of metals at high flow rates. The Ni or Zn uptakes in column sorption were not markedly affected when the flow rates were increased from 80mllh up to 800ml/h for the 5g biomass used. The cation heavy metals removed from waste effluents were recovered in a concentrated solution of small volume. The desorption of bound Ni and Zn ions from the saturated biomass was accomplished with either O.2N HCl or H₂SO₄ that resulted in recoveries of more than 95%. The metals recovered, in the case of Ni and Zn, are identical to that of plating agents ego nickel sulphate or chloride, so that recycling of the metals is possible. An effluent-free, closed loop of Ni or Zn treatment system was proposed, whereby the Ni or Zn ions can be recycled to the plating bath whilst the purified water is fed back to the rinse tanks. Ca and Mg ions, commonly present in the electroplating effluents, appeared to affect sorption of heavy metals by azolla when metal concentrations were relatively low, presumedly through its competitive binding for the shared sites on surfaces of azolla. The data obtained from column sorption of Ni and Zn follows the BDST model well, enabling the application of the model to predicting design parameters for scale-up of the biosorption column system. It is interesting that the values of metal uptake, expressed in molar quantities, obtained on respective single-metal solutions and the multiple metal system, are similar, implying that the mechanisms involved in the sorption of all metal cations are similar and that the binding sites on surfaces of azolla are probably shared by all cation metals. The surface of the biomass provides sites for metal binding estimated in the range of 0.45-0.57mmol/g, based on the current experiments. The biomass has a surface area of 429 m²/g and water retention of 14.3 ml/g. The functional groups on the surface of azolla were partially identified using chemical modification and metal binding comparison. Among the functional groups examined, carboxyl groups, provided by amino acids and polysaccharides, appeared to play an important role in metal cation binding. The infrared spectra of the samples support this conclusion.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The polysaccharides that are present in wine originate from the grapes, the fungi that grow on the grapes and from other microorganisms that come into contact with the must during winemaking. The grape-derived polysaccharides of most concern in winemaking are pectin, glucan and xylan that can be enzymatically degraded by pectinases, glucanases and xylanases, respectively. These are the main structural polysaccharides of the cell wall of the grape cell. Degradation of the cell walls will result in the separation and rupture of the grape cells, and cell wall-bound compounds will be released into the must. Treating the must with pectinase and macerating enzyme preparations can result in an increase in free-flow juice, an improvement in must clarification and filtration, and an increased extraction of phenols and tannins. The tannins that are extracted polymerise with anthocyanins in red wine during ageing, resulting in increased colour intensity and stability. Wine aroma is also influenced by enzyme treatment. The degradation of the cell wall contributes to the release of glycosidically-bound terpene or alcohol precursors from the berries. The hydrolysis of these precursors during fermentation can result in an improvement in aroma. It can thus be seen that it is possible to improve wine quality and processing by supplementing the endogenous enzymes that are present in the fermentation with commercial enzyme preparations. Commercial enzymes are typically crude fungal preparations. The majority of commercial pectinase and glucanase preparations are derived from Aspergillus and Trichoderma, respectively. Since the endogenous polysaccharase activity of Saccharomyces cerevisiae is very limited, the heterologous expression of specific polysaccharase genes in an industrial yeast strain can improve the winemaking process, resulting in a higher quality wine without the addition of expensive commercial enzyme preparations. Since only the desired enzymes are secreted by the recombinant strain, there will be no undesired sideactivities, which can be detrimental to wine quality. Several pectinase-, glucanaseand xylanase-encoding genes, cloned from a variety of organisms, have been expressed successfully in laboratory strains of S. cerevisiae. Attempts have also been made to construct industrial wine yeast strains that express these polysaccharase genes and secrete the encoded enzymes. Fermentation with some of these strains resulted in a decrease in total phenolics and turbidity, an increase in juice extraction, and alterations in the colour and aromatic profile of the resulting wines. In this study, four polysaccharide-degrading, recombinant wine yeast strains were constructed. The endo-β-1,4-xylanase gene, XYN2, and the endo-β-1,4-glucanase gene, end1, were previously cloned from the soft rot fungus Trichoderma reesei and the rumen bacterium Butyrivibrio fibrisolvens, respectively. These genes were subcloned into different expression cassettes which were used to construct the four integration plasmids. The recombinant plasmids contained the following gene cassettes: TEF1P-XYN2-ADH2T (plasmid pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmid pDLG30) ADH1P-MFα1S-end1-TRP5T and ADH2P-XYN2-ADH2T (plasmid pDLG33), ADH1P-MFα1S-end1-TRP5T and YG100PXYN2- ADH2T (plasmid pDLG39). These four plasmids were then separately integrated into the ILV2 locus of the commercial wine yeast strain S. cerevisiae VIN13. Wine was made with the four strains constructed in this study, a pectolytic strain, VIN13[pPPK], a glucanase- and xylanase-secreting strain, VIN13[pEX], an untransformed VIN13 strain, and an untransformed strain with the addition of the commercial enzyme preparation Rapidase EX Colour. Microvinification experiments were carried out on Pinot noir, Ruby Cabernet and Muscat d’Alexandria wines. Fermentation with the polysaccharide-degrading strains resulted in significant improvements in juice extraction, colour intensity and stability, and in alterations in the aromatic profiles of the wines produced. Subject to the approval by the regulatory authorities and eventual consumer acceptance of the use of genetically modified organisms (GMOs) in fermented foods and beverages, it might be required that the GM status of the yeast that is used appears on the label. Currently, there is no robust technique available with which the use of GM yeast can be revealed in a finished wine because the yeast cells and their DNA are removed from or denatured in the wine during filtration and processing. One way with which the undeclared use of a GM yeast in winemaking could be exposed would be to compare the chemical profile of a suspect wine with that of non-GM wine. In order to explore this concept further, a secondary aim of this study was to investigate whether Fourier Transformation Infra Red (FT-IR) spectroscopy coupled with multivariate data analysis could distinguish between wines fermented with transgenic and non-transgenic yeast strains, or between wines fermented with different transgenic strains. The results showed that this method could be used to classify wines fermented with different yeast strains if fermentation with the strain resulted in a unique chemical profile in the resulting wine. This was a preliminary study and these findings were summarised as an addendum to the thesis. / AFRIKAANSE OPSOMMING: Die polisakkariede wat in wyn teenwoordig is, is afkomstig van die druiwe, die swamme wat op die druiwe groei en vanaf ander mikroörganismes wat tydens die wynmaakproses met die mos in aanraking kom. Die belangrikste druifpolisakkariede in wynbereiding is pektien, glukaan en xilaan, wat onderskeidelik deur pektinases, glukanases en xilanases afgebreek kan word. Hierdie is die vernaamste strukturele polisakkariede van ‘n druifsel se selwand. Die afbreking van die selwande veroorsaak dat die druifselle skei en skeur, met die gevolg dat die selwandgebonde verbindings in die mos vrygelaat word. Die behandeling van die mos met pektinase en versappingsensiempreparate kan tot ʼn toename in vry-afloopsap lei, sowel as ʼn verbetering in mosverheldering en -filtrasie en ʼn verhoogde ekstraksie van fenole en tanniene. Die tanniene wat geëkstraheer word, polimeriseer in rooiwyn tydens veroudering, en dit lei tot verhoogde kleurintensiteit en -stabiliteit. Wynaroma word ook deur ensiembehandeling beïnvloed. Die afbreking van die druifselwand dra by tot die vrylating van glikosidiesgebonde terpeen- en alkoholvoorlopers uit die korrels. Die hidrolise van hierdie voorlopers tydens gisting kan lei tot ʼn verbetering van die aroma. Dit is dus duidelik dat dit moontlik is om wynkwaliteit en wynbereiding te verbeter deur die endogene ensieme wat in die gisting teenwoordig is met kommersiële ensiempreparate te supplementeer. Kommersiële ensiempreparate is tipies ongesuiwerde swampreparate. Die meerderheid kommersiële pektinase- en glukanasepreparate word onderskeidelik vanaf Aspergillus en Trichoderma verkry. Aangesien die endogene polisakkaraseaktiwiteit van Saccharomyces cerevisiae baie beperk is, kan die heteroloë uitdrukking van spesifieke polisakkarase-gene in ʼn industriële gisras die wynbereidingsproses verbeter en lei tot ʼn hoër kwaliteit wyn sonder die byvoeging van duur kommersiële ensiempreparate. Omdat die verkose ensieme deur die rekombinante ras uitgeskei word, sal daar geen ongewenste newe-effekte teenwoordig wees wat ʼn nadelige effek op wynkwaliteit kan hê nie. Verskeie mikrobiese gene wat vir pektinases, glukanases en xilanases kodeer, is reeds voorheen uit ‘n wye verskeidenheid van organismes gekloneer en suksesvol in laboratoriumrasse van S. cerevisiae uitgedruk. Pogings is ook aangewend om industriële wyngisrasse te konstrueer wat hierdie polisakkarasegene uitdruk en hul enkodeerde ensieme uitskei. Gisting met sommige van hierdie rekombinante gisrasse het gelei tot ʼn afname in totale fenoliese verbindings en troebelheid, ʼn verhoging in sapekstraksie, en veranderings in die kleur en aromatiese profiel van die gevolglike wyne. In hierdie studie is vier polisakkaried-afbrekende, rekombinante wyngisrasse gekonstrueer. Die endo-β-1,4-xilanasegeen, XYN2, en die endo-β-1,4- glukanasegeen, end1, is voorheen reeds onderskeidelik vanaf die sagte vrotswam, Trichoderma reesei, en die rumenbakterium, Butyrivibrio fibrisolvens, gekloneer. Hierdie gene is in vier integrasieplasmiede in verskillende ekspressiekassette gesubkloneer. Die plasmiede het die volgende geenkassette bevat: TEF1P-XYN2- ADH2T (plasmied pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmied pDLG30) ADH1PMFα1S- end1-TRP5T and ADH2P-XYN2-ADH2T (plasmied pDLG33), ADH1P-MFα1S end1-TRP5T and YG100P-XYN2-ADH2T (plasmied pDLG39). Hierdie vier plasmiede is toe afsonderlik in die ILV2-lokus van die kommersiële wyngisras, S. cerevisiae VIN 13, geïntegreer. Wyn is met hierdie vier gekonstrueerde gisrasse gemaak, die pektolitiese gisras, VIN13[pPPK], die glukanase- en xilanase-afskeidende gisras, VIN13[pEX], die ongetransformeerde VIN13-ras, en met ʼn ongetransformeerde VIN13 gis waarby die kommersiële ensiempreparaat, Rapidase EX Colour, bygevoeg is. Mikro-wynbereidingseksperimente is op Pinot noir-, Ruby Cabernet- en Muscat D’Alexandria wyne uitgevoer. Gisting met die polisakkaried-afbrekende gisrasse het gelei tot ʼn noemenswaardige verbetering in sapekstraksie, kleurintensiteit en kleurstabiliteit, asook in veranderinge in die aromatiese profiele van die geproduseerde wyne. Indien die gebruik van geneties gemodifiseerde organismes (GMOs) in gefermenteerde voedsel en drank deur die reguleringsowerhede goedgekeur en uiteindelik deur die verbruiker aanvaar sou word, sou dit vereis kon word dat die GMstatus van die wyngisgis op die etiket van die wynbottel aangebring word. Verpligte etikettering van GM-wyn sal metodes vereis waarmee die ‘nalentskap’ van GMgisselle in die finale produk geïdentifiseer en gemoniteer kan word. Tans is daar geen robuuste tegnieke beskikbaar waarmee die gebruik van GM-giste openbaar kan word nie, aangesien die gisselle en hul DNA tydens filtrasie en prosessering verwyder word. Een wyse waarop die onverklaarde gebruik van ‘n GM-gis in wynbereiding blootgestel sou kno word, is om die chemiese profiel van die verdagte wyn met dié van ‘n nie-GM-wyn te vergelyk. Ten einde hierdie konsep verder te ondersoek was ‘n sekondêre doelwit van hierdie studie om te bepaal of FT-IR (Fourier-transformasie-infrarooi) spektroskopie tesame met meervariante dataanalise gebruik kan word om te onderskei tussen wyne wat met transgeniese en nietransgeniese gisrasse gegis is, of tussen wyne wat met verskillende transgeniese rasse gegis is. Die resultate het aangedui dat hierdie metode gebruik kan word om wyne wat met verskillende gisrasse gegis is, te klassifiseer indien die betrokke gisras ʼn unieke chemiese profiel in die uiteindelike wyn veroorsaak het. Dit was egter ʼn voorlopige ondersoek en is as ʼn byvoegsel tot die tesis geskryf.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2018 / Beer is one of the most widely consumed alcoholic beverages in the world. The brewing process is based on natural enzymatic activities that take place during the malting of barley grain, mashing of grist and fermentation of wort. Insufficient malt enzyme activity during the mashing process leads to high levels of barley β-glucan, arabinoxylan (AX) and dextrins in the wort as well as in the final beer. It was reported that high levels of β-glucan and AX increase wort and beer viscosity which lower the rate of beer filtration and this negatively affect the production rate in the brewery. During beer fermentation, brewing yeast catalyses the conversion of wort sugars to ethanol, carbon dioxide and other metabolic products. However, non-fermentable carbohydrates i.e., limit dextrins remain in the wort and final beer. These non-fermentable carbohydrates are known to contribute to the caloric value of beer which might lead to weight gain in consumers. The objectives of this study were to evaluate the effect of recombinant yeast strains expressing an endo-β-1,4-glucanase or an endo-β-1,4-xylanase on beer viscosity (as an indicator of filterability) and an α-amylase on residual sugars levels. The effect of the above mentioned enzymes on the aroma, appearance, flavour, mouth-feel and overall quality of the beer was also determined. Wort was produced in the University of Limpopo micro-brewery and the wort was pitched with different recombinant strains. The wild-type strain served as control. The results obtained showed that the xylanase expressing strain produced a measurable decrease in viscosity over the course of the fermentation, but endo-glucanase did not have any effect on the beer viscosity. The α-amylase producing strain, did not show a measurable reduction of residual sugars in the final beer probably as a result of very low activity on α-1,6 glycosidic bonds in dextrins during fermentation. The xylanase and α-amylase producing strain fermented effectively with good attenuation (decrease in wort specific gravity). The beer produced by the α-amylase and control strains were preferred in terms of taste and had similar qualities. The secreted amylolytic activity was not sufficient to significantly reduce residual sugar in the final beer. Although the xylanase secreting strain produced a beer with lower viscosity, the enzyme had a negative impact on the taste of the beer. Key words: Brewer’s yeast, beer fermentation, low calorie beer, amylase, xylanase, endo-glucanase.