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

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with tartaric acid, are natural constituents of grape must and wine, and can be released as free acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms present in wine. These volatile phenols contribute to the aroma of the wine. The Bretfanomyces yeasts are well known for their ability to form volatile phenols in wine. However, these species are associated with the more unpleasant and odorous formation of the ethylphenols and the formation of high concentrations of volatile phenols. Other organisms, including some bacterial species, are responsible for the formation of volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the organoleptic properties of the wine. The enzymes responsible for the decarboxylation of phenolic acids are called phenolic acid decarboxylases; and several bacteria and fungi have been found to contain the genes encoding these enzymes. The following genes have been characterised: PAD1 from Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified. S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast. Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not show any higher enzyme activity than the control strain. Both bacterial genes, under the control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the SMR1 gene as selective marker. The cloning and transformation of pdc and padc into industrial wine yeast strains can therefore be used to detect the effect of phenolic acid decarboxylase genes in the winemaking process for the possible improvement of wine aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1 disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile phenols in the wine. The manipulated strains also produced enzymes capable of producing large amounts of favourable monoterpenes in the wine. This study paves the way for the development of wine yeast starter culture strains for the production of optimal levels of volatile phenols, thereby improving the sensorial quality of wine. / AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige fenole bekend en kan tot die aroma van wyn bydra. Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die 4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n aanvullende effek op die wyn aroma kan hê. . Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer. S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag. Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen (PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is. Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene, onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In verhoogde mate gedurende hierdie proses gevorm. Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van die wyn te verbeter.

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry. / National Research Foundation

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid environment. This aggregation of cells, also referred to as "floc formation", will in most cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast strains, the floes can move to the surface of the liquid growth substrate to form a thin layer, called a "velum", that has been compared to other microbial biofilms. The factors that trigger flocculation can be divided into two groups, physical/chemical (e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that are either directly or indirectly involved in flocculation). In top-fermenting yeast strains, several physical and chemical factors that trigger the process have been described, including ethanol concentration, the presence of organic solvents, the absence of molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors, no specific genes have thus far been associated with flocculation in top fermenting yeast strains. In bottom-fermenting yeast strains, the physical and chemical factors that affect the process are similar to the ones described for top-fermenting yeast strains, but include, more specifically, the concentration of hexoses in the media (mannose or glucose), which may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type (inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various genes have been associated with the flocculation of bottom-fermenting yeast strains. Through genetic analysis, the genes have been categorised into dominant genes, semidominant genes and recessive genes. In order to better understand the role of some of the proteins responsible for flocculation in S. cerevisiae, and to create strains whose flocculation properties would correspond to those wanted in the wine and beer industries, three of the dominant flocculation genes, FL01, FL05 and FL011, were placed under the control of the promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved by replacing the native promoters of the flocculation genes with the heterologous promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent due to the absence of the transcription factor that is required for flocculation, F108p,was used as a model system. Some of the transformed strains showed high flocculation, especially when the genes were placed under control of the ADH2 promoter. In addition to this, the strains carrying a modified FL011 gene showed increased adhesion to solid agar media and were able to invade the growth substrate. These strains also showed an increased velum-forming ability when grown in media containing only non-fermentable carbon sources. / AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie, en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste, beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie te vorm. Hierdie verskynsel was ook al gevind in ander organismes. Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie, die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie, pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie. Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose), wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en ressessiewe flokkulerende gene. Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë, was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was verkry deur die vervanging van die wilde tipe promotors van die drie gene met die stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van 'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike gene in flokkulasie, was gebruik as 'n wilde tipe ras. Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om 'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare koolstofbronbevattende media opgegroei word.

The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.

The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.

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.