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.

Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps

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.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. / Filamentous fungi have been employed for production of heterologous proteins such as enzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslational modifications of these proteins. With an improvent in recombinant DNA technologies it has become possible to express many useful proteins in species such as the Aspergilli. However the submerged cultivation of filamentous fungi is complicated by the difficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluids that result. The purpose of the project was to investigate the potential of simultaneous control of morphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, in order to overcome problems associated with the submerged cultivation of filamentous fungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in a filamentous form or a yeast-like morphology in response to environmental conditions. Recently, advances were made in transformation of Mucor, and it has become possible to transform M. circinelloides to express heterologous proteins. The first example of a strong, regulated promoter from M. circinelloides being used for recombinant protein production was the expression of the glucose oxidase gene (from Aspergillus niger) under the control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucose oxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, to produce low-kilojoule beverages and to measure glucose levels in medical diagnostic applications. The scope of this project was to establish the conditions for yeast and filamentous growth of M. circinelloides in order to allow control of morphology, and to evaluate enzyme production under these conditions. Enzyme production of the GOX producing mutant strain, that was recently constructed, was compared to that of a wild type M.circinelloides strain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage and then a filamentous stage. The yeast morphology was induced by anaerobic conditions while the filamentous morphology was achieved by exposure to air. The enzyme, biomass and metabolite production of the glucose-oxidase producing mutant strain and the wild type were monitored during the two-stage fermentations. GOX from the mutant and native amylase activity levels from the wild type were compared to each other and to other production systems for these enzymes. The morphology could be maintained in a yeast form under N2 with addition of ergosterol and Tween 80. The GOX activity levels in the culture fluid were comparable to some of the unoptimized GOX production systems in literature, but much lower than the optimized, recombinant GOX production systems that employ certain yeasts, or Aspergilli or Penicillium. The intracellular GOX levels were almost 6-fold higher than the extracellular levels which was unexpected as GOX is usually well-secreted. The morphological control improved the morphology for the initial yeast-stage of the fermentation but did not improve the morphology during the filamentous, enzyme-producing stage and it decreased the biomass yield and enzyme production by 50%. The constraint of Mucor to its yeast-like form did not improve the broth homogeneity or enzyme production and increased the time required for enzyme production. In this study M. circinelloides did not perform that well against other species already used to produce these enzymes. However, M. circinelloides could be used to produce enzymes from zygomycetes that systems such as A. niger do not produce well.

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 (MScFor)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Unextracted (control) and PHWe Eucalyptus grandis wood chips were pulped at 15% active alkali (AA) and 1% antraquinone (AQ). Another batch of wood chips were then inoculated with fungal co-cultures of Aspergillus flavipes and Pycnoporus sanguineus. FCCi wood chips were incubated for four weeks; one PHWe inoculated experimental treatment was incubated for three weeks. The full pulping cycle (160 min) was used to digest the experimental treatments with the exception of one lot of PHWe wood chips that were pulped for 150 minutes. A further experimental treatment of PHWe wood chips was cooked at a reduced AA charge of 14% and 1% AQ. Analysis of variance (ANOVA) of the data from all the experimental treatments was conducted and the differences within the experimental treatments were determined using Statistica (v7, 1984–2006). The F-value (Fischer distribution) and the p-value as well as a non-parametric test known as the Mann-Whitney procedure was tested at the 95% confidence limit. For a further enhancement of the 95% confidence limit the screened yield data was tested by the Bootstrap method. Scanning electron micrographs clearly demonstrated the changed structure and appearance of the chip cross-sectional area after the different pretreatments. Although the mean average results of all the screened pulp yields showed no significant statistical difference (p> 0.05), differences in screened yield of up to 2.5% were obtained. All the weighted means of the rejects showed a significant difference (p < 0.05). Other pulp properties like shive content, chemical consumption, Kappa number, handsheet brightness and strength tests showed mixed results i.e. rejected or accepted the hypothesis (p> or =or < 0.05). The hypothesis that the combined PHWE and FCCI of wood chips would further increase the pulp yield had to be rejected. It is however anticipated that the combination of PHWE with successive co-culture fungal pretreatment would be very beneficial in obtaining higher pulp yields for fully bleached chemical pulp. Further research would be required to test this assumption. This investigation confirmed the expected beneficial effects of combined PHWE and FCCI pretreatments of wood chips on the strength properties. In addition the combined treatment also improved the initial bonding strength potential of the unbeaten fibres. / AFRIKAANSE OPSOMMING: Onbehandelde en met onder druk, warm water uitgeloogde Eucalyptus grandis houtspaanders is respektiefwelik met 15% aktiewe alkali (AA) en 1% antrakinoon (AQ) verpulp. Hierdie is dan met swamkokulture van Aspergillus flavipes en Pycnoporus sanguineus inokuleer en respektiewelik vir drie en vier weke inkubeer. Onder druk uitgeloogde houtspaanders is ook vir 150 minute verpulp by 15% AA 1% AQ en by ‘n verminderde AA van 14%. Pulpevaluasies is uitgevoer op alle eksperimentele behandelinge. Alle onder druk uitgeloogde en met swamkokultuur inokuleerde houtspaanders het ‘n laer pulpopbrengs, uitskot, skilferinhoud, Kappanommer en ‘n hoër RAA en helderheid opgelewer in vergelyking met die vars houtspaanders. Die vars en warm water uitgeloogde houtspaanders het soortgelyke pulpopbrengs opgelewer. ‘n Variansieanalise (ANOVA) van die data van alle eskperimentele behandelings is uitgevoer gebruikmakende van Statistica (V7, 1984 – 2006). Die F-waarde (Fischer-verspreiding) an die p-waarde so wel as ‘n parametriese toets (Mann-Whitney prosedure) is getoets by ‘n 95% betroubaarheidsgrens. Vir ‘n verdere verhoging van die 95% betroubaarheidsgrens van die pulpopbrengs, is die beskikbare data weer getoets met die Bootstrap-metode. Alle gemiddelde pulpopbrengswaardes het geen beduidende statistiese verkil opgelewer nie (p>0.05), alhoewel verskille van tot 2.5% in pulpopbrengs verkry is. Alle gemiddelde uitskotwaardes het ‘n beduidende verskil getoon (p<0.05). Die ander pulpeienskappe soos skilferinhoud, verbruik aan chemikalieë, Kappagetal, handvel helderheid en sterktewaardes het gemengde resultate opgelewer maw verwerping of aanvaarding van die hipotese p> or =or < 0.05. Die hipotese dat die gekombineerde PHWE en FCCI van die houtspaanders die pulpopbrengs verder sou verhoog moes verwerp word. Daar word egter verwag dat die kombinasie van PHWE met opeenvolgende swamkokultuur behandeling baie voordelig sou wees op die pulpopbrengs van ‘n ten volle gebleikte chemiese pulp. Verdere navorsing is nodig om hierdie veronderstelling te toets. Die ondersoek het die verwagte woordelige effek van die gekombineerde PHWE en FCCI voorbehandelings van die houtspaanders op die papierstrkte-eienskappe bevestig. Bo en behalve dit, het die gekombineerde behandeling ook die aavanklikte bindsterkte potensiaal van die ongeklopte vessels verbeter.

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.