Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Published Article / The growth of Saccharomyces cerevisiae strain W303-1A(a) was evaluated in complex and chemically defined media. The use of chemically defined medium allowed the complete utilisation of glucose within 20 h. as well as all of the produced ethanol within 45 h. Maximum specific growth rates (µmax) were increased from 0.28 h-1 to 0.42 h-1 and the volumetric rate of ethanol production increased from 0.204 g l-1 h-1 to 0.597 g l-1 h-1. However, when the ethanol concentration exceeded a threshold value of 10 g l-1, the µmax value was significantly decreased. These observations suggest that ethanol metabolism related growth experiments for the relevant strain should be carried out in chemically defined medium with ethanol concentrations below 10 g l-1.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: L-Carnitine is a unique and important compound in eukaryotic cells. In Saccharomyces cerevisiae, L-carnitine plays a role in the transfer of acetyl groups from the peroxisomes to the mitochondria. This takes place with the help of the carnitine acetylcarnitine shuttle. The activated acyl group of acetyl-CoA in the peroxisome is transferred to carnitine with the help of a peroxisomal carnitine acetyltransferase to form an acetylcarnitine ester, releasing the CoA-SH. This ester is then transported through the peroxisomal membrane to the cytosol from where it is transported to the mitochondrion. After transport of the acetylcarnitine through the mitochondrial membranes, the reverse reaction takes place in the matrix with the help of a mitochondrial carnitine acetyltransferase, releasing carnitine and the acyl group. In S. cerevisiae, the main carnitine acetyltransferase contributing to >95% of the total carnitine acetyltransferase activity, is encoded by a single gene, CAT2. Cat2p has a peroxisomal and mitochondrial targeting signal and is located to the peroxisomal membrane and the inner-mitochondrial membrane, respectively. The reason for the activated acyl group to be transferred in the form of an acetylcarnitine, is that the peroxisomal membrane is impermeable to acetyl-CoA. This means that the acyl group needs to be transported in the form of intermediate compounds. Acetyl-CoA is formed in the peroxisome of S. cerevisiae as a result of p-oxidation of fatty acids. In yeast, the peroxisome is the sole site for p-oxidation. Fatty acids are transported to the peroxisome where they are oxidized by the p-oxidation cycle to form two-carbon acyl groups in the form of acetyl-CoA. These two-carbon acyl groups are then transferred from the peroxisome to the rest of the cell for gluconeogenesis and other anabolic pathways, or used in the tricarboxylic acid cycle (TCA) of the mitochondia to generate ATP. In this way, it is possible for the cell to use fatty acid as a sole carbon source. There is a second pathway allowing for the utilization of activated acyl groups produced in the peroxisome and that is the glyoxylate cycle. The glyoxylate cycle is a modified TCA cycle, which results in the synthesis of C4 succinate from two molecules of acetyl-CoA. In S. cerevisiae, all of the enzymes of the glyoxylate cycle are located in the peroxisome except for one, whereas in other yeasts studied, all of the glyoxylate enzymes are peroxisomal. As a result of the glyoxylate cycle, the two carbons of acetyl-CoA can leave the peroxisome in the form of succinate or other TCA intermediates like malate and citrate. These compounds are transferred through dicarboxylic acid carriers present in the peroxisomal membrane and used in further metabolic needs of the cell. To understand the role of carnitine in the cell, a strategy for the cloning of genes involved in carnitine-dependent activities in S. cerevisiae was developed. The disruption of the citrate synthetase gene, CIT2, of the glyoxylate cycle yielded a strain that was dependent on carnitine when grown on the fatty acid oleic acid. This allowed for a mutagenesis strategy based on negative selection of mutants affected in carnitine-dependent activities. The ~cit2 strain was mutagenized and plated on minimal media. After replica plating on oleic acid media, mutant strains were selected that were unable to grow even in the presence of carnitine. In order to eliminate strains with defects in peroxisome biogenesis and ~-oxidation, and only select for strains with defects in carnitine-dependent activities, the mutant strains were transformed with the CIT2 gene to restore the glyoxylate cycle. Mutants that grew on oleic acid after transformation, and which are therefore not affected in activities independent of carnitine, were retained for further analysis. Transforming one of these mutants with a S. cerevisiae genomic library for functional complementation, yielded a clone carrying the YAT1 gene, coding for the carnitine acetyltransferase of the outer-mitochondrial membrane. No phenotype had previously been assigned to a mutant allele of this gene. / AFRIKAANSE OPSOMMING: L-Karnitien is 'n unieke en belangrike verbinding in eukariotiese selle. In Saccharomyces cerevisiae speel L-karnitien In rol in die oordrag van asielgroepe van die peroksisoom na die mitochondrion. Dit vind plaas met behulp van die karnitien-asetielkarnitien-weg. Die geaktiveerde asiel groep van asetiel-KoA in die peroksisoom word na karnitien oorgedra met behulp van 'n peroksisomale karnitien-asetielkarnitien-transferase-ensiem om 'n asetielkarnitien ester te vorm, waarna die KoA-SH vrygestel word. Hierdie ester word dan deur die peroksisomale membraan na die sitoplasma vervoer waarna dit na die mitochondrion vervoer word. Nadat die asetielkarnitien deur die mitochondriale membrane vervoer is, vind die omgekeerde reaksie in die matriks plaas met behulp van die mitochondriale karnitien-asetielkarnitien-transferase-ensiem, waarna die karnitien en die asielgroep vrygestel word. In S. cerevisiae word die hoof karnitien-asetielkarnitien transferase wat tot >95% van die totale karnitien-asetielkarnitien-transferase-aktiwiteit bydra, deur 'n enkele geen, CA T2 gekodeer. CAT2p het 'n peroksisomale en mitochondriale teikensein en dit word onderskeidelik na die peroksisomale en binnemitochondriale membrane gelokaliseer. OPSOMMING Die geaktiveerde asielgroep word in die vorm van 'n asetielkarnitien vervoer omdat die peroksisomale membraan ondeurlaatbaar vir asetiel-KoA is. Dit beteken dat die asielgroepe slegs in die vorm van intermediêre verbindings vervoer kan word. Asetiel-KoA word weens p-oksidasie van vetsure in die peroksisoom van S. cerevisiae gevorm. In gis is die peroksisoom die enigste plek waar p-oksidasie plaasvind. Vetsure word na die peroksisoom vervoer waar dit deur die p-oksidasiesiklus geoksideer word om tweekoolstof asielgroepe in die vorm van asetiel-KoA te vorm. Hierdie twee-koolstof asielgroepe word dan vanaf die peroksisoom na die res van die sel vervoer vir glukoneogenese en ander metaboliese paaie, of dit word in die trikarboksielsuursiklus (TKS) van die mitochondrion gebruik om ATP te genereer. Op hierdie wyse is dit moontlik vir die sel om vetsure as enigste koolstofbron te benut. Die glioksilaatsiklus is 'n tweede weg wat die benutting van asielgroepe, wat in die peroksisoom geproduseer is, toelaat. Die glioksilaatsiklus is 'n gemodifiseerde TKS-siklus wat die sintese van C4 suksinaat van uit twee molekules asetiel-KoA bewerkstellig. In teenstelling met ander giste waar al die glioksilaatsiklus ensieme in die peroksisoom geleë is, kom een van S. cerevisiae se ensieme buite die peroksisoom voor. Die resultaat van die glioksilaatsiklus is dat die twee koolstowwe van asetiel-KoA die peroksisoom in die vorm van suksinaat of ander TKS-intermediêre verbindings soos malaat en sitraat, kan verlaat. Hierdie verbindings word deur middel van dikarboksielsuur-transporters in die peroksisomale membraan vervoer en word dan vir verdere metaboliese behoeftes in die sel gebruik. Om die rol van karnitien in die sel te verstaan, is 'n strategie ontwikkel om gene wat by karnitien-afhanklike aktiwiteite in S. cerevisiae betrokke is, te kloneer. Die disrupsie van die sitraatsintesegeen, CIT2, van die glioksilaatsiklus het 'n ras gelewer wat van karnitien vir groei op die vetsuur oleiensuur afhanklik was. Die fl.cit2-ras is gemuteer en op minimale media uitgeplaat. Na replika-platering op oleiensuur media, is mutante rasse geselekteer wat nie gegroei het nie, selfs nie in die teenwoordigheid van karnitien nie. Om mutantrasse uit te skakel wat defekte in peroksisoom-biogenese en p-oksidasie het en net mutantrasse te selekteer wat defekte in karnitien-afhanklike aktiwiteite het, is die rasse met die CIT2- geen getransformeer om die glioksilaatsiklus te herstel. Mutante wat na transformasie op oleiensuur gegroei het, en dus nie in aktiwiteite onafhanklik van karnitien geaffekteer is nie, is behou en aan verdere analise blootgestel. Komplimentering van een van hierdie mutante met 'n S. cerevisiae genomiese biblioteek, het 'n kloon wat die geen YAT1 bevat, gelewer. YAT1 is 'n geen wat die karnitienasetieltransferase van die buite-mitochondriale membraan kodeer. Geen fenotipe is ooit voorheen aan 'n mutant alleel in hierdie geen toegeskryf nie.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: L-carnitine is a compound with a long history in biochemistry. It plays an important role in mammals, where many functions have been attributed to it. Those functions include the p-oxidation of long-chain fatty acids, the regulation of the free CoASH/ Acyl-CoA ratio and the translocation of acetyl units into mitochondria. Carnitine is also found in lower eukaryotic organisms. However, in contrast to the multiple roles it plays in mammalian cells, its action appears to be restricted to the transport of activated acyl residues across intracellular membranes in the lower eukaryotes. In the yeast Saccharomyces cere visiae , the role of carnitine consists mainly of the transfer of activated acetyl residues from the peroxisome and cytoplasm to the mitochondria. This process is referred to as the carnitine shuttle. This system involves the transfer of the acetyl moiety of acetyl-CoA, which cannot cross organellar membranes, to a molecule of carnitine. Subsequently, the acetylcarnitine is transported across membranes into the mitochondria, where the reverse transfer of the acetyl group to a molecule of free CoA occurs for further metabolism. Carnitine acetyl transferases (CATs) are the enzymes responsible for catalysing the transfer of the activated acetyl group of acetyl-CoA to carnitine as well as for the reverse reaction. In the yeast S. cerevisiae, three CAT enzymes, encoded by the genes CAT2, YAT1 and YAT2, have been identified. Genetic data suggest, that despite the high sequence similarity, each of the genes encodes for a highly specific activity that is part of the carnitine shuttle. So far, the specific function of any of the three CAT enzymes has been elucidated only partially. The literature review focuses mainly on the importance of the carnitine system in mammals. After discussing the discovery and biosyntheses of carnitine, the enzymatic background of and molecular studies on the carnitine acyltransferases are described. The experimental section focuses on elucidating the physiological roles and cellular localisation of the three carnitine acetyltransferase of S. cere visia e. We developed a novel enzymatic assay to study CAT activity in vivo. By C-terminal tagging with a green fluorescent protein, we localised the three CAT enzymes. However, all our genetic attempts to reveal specific roles for and functions of these enzymes were unsuccessful. The overexpression of any of the CAT genes could not cross-complement the growth defect of other CAT mutant strains. No phenotypical difference could be observed between strains carrying single, double and triple deletions of the CAT genes. Furthermore, the expression of the Schizosaccharomyces pombe dicarboxylic acid transporter can complement the deletion of the peroxisomal citrate synthase, but has no effect on the carnitine shuttle per se. Our data nevertheless suggest that Cat2p is the enzyme mainly responsible for the forward reaction, e.g. the formation of acetylcarnitine and free CoA-SH from acetyl-CoA and carnitine, whereas Yat1 pand Yat2p may be required mainly for the reverse reaction. / AFRIKAANSE OPSOMMING: L-karnitien is 'n verbinding met 'n lang geskiedenis in die biochemie-veld. Dit speel 'n belangrike rol in soogdiere, waar verskeie funksies daaraan toegeskryf word. Dié funksies sluit in die p-oksidasie van lang-ketting-vetsure, die regulering van die vrye KoA-SH-tot-asiel-KoA-verhouding en die oordrag van asetieleenhede na die mitochondria. Karnitien word ook in laer eukariotiese organismes gevind. In teenstelling met die verskeidenheid rolle wat dit in soogdierselle vervul, is die funksie in laer eukariote tot die transport van geaktiveerde asetielderivate oor intrasellulêre membrane beperk. In die gis Saccharomyces cerevisiae is die funksie van karnitien meestal beperk tot die vervoer van geaktiveerde asetielresidu's vanaf die sitoplasma en piroksisome na mitochondria, 'n proses wat as die "karnitiensiklus" bekend staan. Die proses behels die oordrag van die asetielgedeelte van asetiel-KoA, wat nie oor organelmembrane kan beweeg nie, na 'n molekuul van karnitien. Gevolglik word die asetielkarnitien oor die membraan na die mitochondria vervoer, waar - met die oog op verdere metabolisme - die omgekeerde oordrag van die asetielgroep na 'n vrye molekuul van KoA plaasvind. Karnitienasetiel-transferases (KAT's) is die ensieme wat verantwoordelik is vir die katalisering van die oordrag van die geaktiveerde asetielgroepe van asetiel-KoA na karnitien, sowel as vir die omgekeerde reaksie. In die gis S. cerevisiae is drie KAT-ensieme geïdentifiseer wat deur die gene CAT2, YAT1 en YAT2 gekodeer word. Genetiese data dui daarop dat, ten spyte van die hoë mate van homologie van die DNA-volgordes, elke geen vir 'n hoogs spesifieke aktiwiteit, wat deel van die karnitiensiklus is, kodeer. Tot dusver is die spesifieke funksie van die drie individuele KAT-ensieme net gedeeltelik ontrafel. Die literatuurstudie fokus hoofsaaklik op die belangrikheid van karnitiensisteme in soogdiere. Na 'n bespreking van die ontdekking en biosintese van karnitien, word die ensimatiese agtergrond en molekulêre studies van KAT's beskryf. Die eksperimentele deel konsentreer op die ontrafelling van die fisiologiese rol en intrasellulêre lokalisering van die drie KAT-ensieme van S. cerevisiae. Eerstens is 'n nuwe ensimatiese toets ontwikkel om KAT-aktiwiteit in vivo te bestudeer. Deur C-terminale aanhegting van 'n groen fluoreserende proteïen kon die drie KATensieme gelokaliseer word. Daar kon egter nie met behulp van genetiese studies verder lig gewerp word op die spesifieke rolle en funksies van hierdie KAT-ensieme nie. Die ooruitdrukking van enige van die KAT-gene kon nie die groeidefek van ander KAT-mutantrasse kruiskomplementeer nie. Geen fenotipiese verskil tussen rasse wat 'n enkel, dubbel of trippel delesie van die KAT-gene bevat, kon waargeneem word nie. Verder kon die uitdrukking van Schizosaccharomyces pombe se dikarboksielsuurtransporter die delesie van die peroksisomale sitraatsintetase komplementeer, maar het dit as sulks geen effek op die karnitiensiklus gehad nie. Die data wat deur hierdie studie verkry is, dui nogtans daarop dat Cat2p die ensiem is wat hoofsaaklik verantwoordelik is vir die voorwaartse reaksie, met ander woorde die vorming van asetielkarnitien en vrye KoH-SH van asetiel-KoA en karnitien, terwyl Yat1 p en Yat2p hoofsaaklik vir die omgekeerde reaksie benodig word.

Thesis (DSc (Microbiology))--University of Stellenbosch, 2005. / The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.

published_or_final_version / Botany / Master / Master of Philosophy

No description available.

In eukaryotes, chromosomes are non-randomly positioned within the nucleus.  The perinuclear localization of <i>S. cerevisiae </i>telomeres provides a useful model for studying mechanisms that control chromosome positioning.  In budding yeast, telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus.  In this thesis, I investigate whether DNA replication causes telomere dislodgment from the nuclear periphery. First, using live-cell fluorescence microscopy I show that delaying DNA replication causes a corresponding delay in the dislodgement of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization.  Second, I show that telomere dislodgment is not simply the result of recruitment of telomeres to a replication factory that is formed in the nuclear interior, since I found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior.  The telomere binding complex Ku is one of the factors that establishes telomere localization to the nuclear envelope.  Using a gene locus tethering assay,  I show that the Ku-mediated telomere localization pathway is inactivated after DNA replication. Based on these findings, I propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway.  In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.

No description available.