Dissertations / Theses on the topic 'Saccharomyces cerevisiae – Genetic aspects'

For organisms undergoing a developmental process it is ideal that specific genes are induced and repressed at the correct time and to the correct level in a coordinated manner. The process of meiosis and spore formation (collectively known as sporulation) in Saccharomyces cerevisiae provides a convenient system to elucidate transcriptional mechanisms of gene repression and the contribution such repression mechanisms offer to cells capable of undergoing a developmental process. This thesis focuses on transcriptional repression of sporulation-specific genes during both vegetative/mitotic conditions and sporulation. The fitness contribution of transcriptional repressors that regulate sporulationspecific genes during vegetative growth were investigated considering the similarities between meiosis and mitosis such as DNA replication, chromosome segregation and cytokinesis. Well-characterised sporulation genes of different functions were expressed in vegetative cells and ectopic expression of these genes was found not to be lethal. It was ascertained through strain competition studies that ectopic expression of the genes IME1, SMK1, SPR3 and DIT1 during mitotic growth did not affect cellular fitness. The expression of NDT80 in vegetative cells, however, caused a marked reduction in fitness and cells were also further compromised in the absence of the Sum1p repressor that regulates NDT80 transcription. The role of NDT80 as a transcriptional activator of middle sporulation genes, rather than the over-expression of NDT80 as a protein, caused the reduction of cell viability. Transcriptional regulation of the middle sporulation-specific gene SPR3 by the meiosis-specific Set3p repressor complex was investigated using synchronous sporulation cultures of the W303a/?? strain commonly used for sporulation studies. In a mutant W303a/?? ??set3/??set3 strain, lacking a key component of the Set3p repression complex, the transcription of SPR3 was uncharacteristically expressed at higher levels and derepressed during late sporulation. This SPR3 expression was consistent for both SPR3 transcript and SPR3::lacZ reporter protein studies. This preliminary work will enable future studies, using SPR3 promoter deletions fused to a lacZ reporter, aimed at determining the region of the SPR3 promoter that the Set3p complex may interact with to transcriptionally repress the gene during sporulation.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.
Includes bibliographical references.
Aging is a universal process that affects organisms from yeast to humans. Replicative life span in the budding yeast, Saccharomyces cerevisiae is defined as the number of daughter cells produced by a mother cell prior to senescence. The isolation and characterization of genes and interventions that extend mother cell life span can provide insight into the mechanisms of aging. One cause of aging in yeast is the accumulation of extrachromosomal ribosomal DNA circles (ERCs) in the mother cell nucleus. ERCs are formed by homologous recombination within the ribosomal DNA (rDNA) caused by the presence of a stalled replication fork. Mutation of the replication fork block protein Foblp dramatically reduces ERCs and extends life span. A central regulator of longevity in yeast is the silencing protein Sir2p. Deletion of SIR2 shortens life span and overexpression of SIR2 extends life span. Sir2p promotes silenced chromatin at the rDNA by catalyzing a novel NAD-dependent histone deacetylation reaction. This rDNA silencing function is likely to promote long life span by inhibiting rDNA recombination and, hence, the formation of ERCs. Sir2p is required for life span extension by caloric restriction (CR), demonstrating the important role that this protein plays in the aging process. CR is thought to activate Sir2p by increasing the amount of NAD that is available as a substrate for Sir2p. The finding that osmotic stress extends life span by a mechanism that genetically mimics CR supports this. High osmolarity causes a metabolic shift from fermentation to an NAD-generating glycerol biosynthesis pathway.
(cont.) Life span extension by high osmolarity requires both Sir2p and glycerol biosynthesis. SSD1-V defines the only known Sir2p independent pathway that promotes long life span. SSD1-V functions in many different cellular processes and the mechanism(s) by which it extends life span is not known. SSDI-V functions in a pathway parallel to the longevity promoting protein Mpt5p for cell integrity and interacts genetically with the aging gene UTH1 in several, apparently unrelated, cellular processes. Further defining the molecular nature of this Sir2p-independent longevity pathway will provide insight into the aging process in yeast and, perhaps, higher organisms as well.
by Matt Kaeberlein.
Ph.D.

The neurodegenerative disorders Alzheimer??s Disease (AD) and Parkinson??s Disease (PD) are characterised by the accumulation of misfolded amyloid beta 1-42 peptide (Aβ1-42) or α-synuclein, respectively. In both cases, there is extensive evidence to support a central role for these aggregation-prone molecules in the progression of disease pathology. However, the precise mechanisms through which Aβ1-42 and α-synuclein contribute to neurodegeneration remain unclear. Organismal, cellular and in vitro models are under development to allow elucidation of these mechanisms. A cellular system for the study of intracellular Aβ1-42 misfolding and localisation was developed, based on expression of an Aβ1-42-GFP fusion protein in the model eukaryote Saccharomyces cerevisiae. This system relies on the known inverse relationship between GFP fluorescence, and the propensity to misfold of an N-terminal fusion domain. To discover cellular processes that may affect the misfolding and localisation of intracellular Aβ1-42, the Aβ1-42-GFP reporter was transformed into the S. cerevisiae genome deletion mutant collection and screened for fluorescence. 94 deletion mutants exhibited increased Aβ1-42-GFP fluorescence, indicative of altered Aβ1-42 misfolding. These mutants were involved in a number of cellular processes with suspected relationships to AD, including the tricarboxylic acid cycle, chromatin remodelling and phospholipid metabolism. Detailed examination of mutants involved in phosphatidylcholine synthesis revealed the potential for phospholipid composition to influence the intracellular aggregation and localisation of Aβ1-42. In addition, an existing S. cerevisiae model of α-synuclein pathobiology was extended to study the effects of compounds that have been hypothesized to be environmental risk factors leading to increased risk of developing PD. Exposure of cells to aluminium, dieldrin and compounds generating reactive oxygen species enhanced the toxicity of α- synuclein expression, supporting suggested roles for these agents in the onset and development of PD. Expression of α-synuclein-GFP in phosphatidylcholine synthesis mutants identified in the Aβ1-42-GFP fluorescence screen resulted in dramatic alteration of α-synuclein localisation, indicating a common involvement of phospholipid metabolism and composition in modulating the behaviours of these two aggregation-prone proteins.

The brewing properties of an industrial strain of Saccharomyces cerevisiae were investigated by laboratory scale brewing trials in the presence or absence of an uncoupler of oxidative phosphorylation (SX-1). When SX-1 was added the change in specific gravity of brewing wort with respect to time was less, yeast produced less biomass and more ethanol per unit drop in specific gravity than the control. Similar fermentation properties were observed for C2, a haploid laboratory yeast strain with the ability to ferment maltose. Recombinant DNA technology was used to generate a C2 pet mutant, specifically in the ATP12 gene, which encodes a protein essential for mitochondrial ATP synthesis. In brewing trials a comparison of C2 and C2:AATP 12 shows similar results to C2 fermentations in the presence or absence of SX-1 although the effects of SX-1 are more dramatic than with C2:AA TP 12. Together, the results of chemical studies and gene disruption mutagenesis suggest that mitochondrial ATP synthesis affects nuclear functions, although the possibility that results obtained are a consequence of changes to the yeast mitochondrial DNA cannot be eliminated. Problems experienced with yeast DNA transformation protocols lead to the development of a new transformation method that is quicker and more efficient than the standard protocol. Initial studies revealed that DMSO could enhance yeast transformation efficiency, and that the optimal concentration of DMSO used is strain specific. The point at which DMSO was added was found to be important, with maximal transformation efficiency achieved when DMSO was added just before heat shocking. The optimised protocol for S. cerevisiae JRY188 routinely enhanced transformation IS- to 25-fold compared with a control transformation protocol. The osmotic condition was found to be important for DNA uptake as transformation was inhibited if yeast were washed in 1M sorbitol and selected on plates containing the same. Significantly, DMSO enhanced transformation even in the absence of captions, therefore this method may prove useful for yeasts which transform poorly by existing cationic-based yeast transformation methods.

A genetic analysis of thiamine metabolism has been carried out in the budding yeast, Saccharomyces cerevisiae. A collection of thiamine auxotrophic mutants were isolated following UV and Ty insertion mutagenesis. The mutations responsible for the auxotrophic phenotypes were characterised to different extents through complementation analysis, molecular cloning and enzyme assays. In total 171 mutants were analysed and all of these have been assigned to complementation groups, genes and/or functions. Some newly isolated mutations were found to be allelic with the known biosynthetic genes, THI4 and THI6 others were in the regulatory genes, THI2 and THI3 two more defined a new function for the transcription factor, Pdc2p, namely thiamine gene activation. In addition the previously known mutations, thil, thi2, and thi3, were complemented and the sequences of the wild-type THI1, THI2 and THI3 genes were found. From the deduced amino acid sequences roles for the gene products were hypothesised. The Thi2p was found to be homologous with the Gal4p transcription factor due to the presence of a Zn-finger motif therefore a DNA-binding transcription factor role was proposed for this protein. The Thi3p was found to be homologous to the structural proteins for the enzyme pyruvate decarboxylase. It contains a conserved sequence for TPP binding, the consensus motif having been found in all TPP-dependent enzymes. Therefore it is hypothesised here that Thi3p acts as a "TPP sensor" within the cell, such that deactivation of thiamine-regulated genes is exerted when TPP is bound to Thi3p. In the case of THI1, the complementing ORF was found to be a previously characterised gene, ILV2, which encodes the aceto-hydroxy acid synthase (AHAS) enzyme. AHAS catalyses the first step in the parallel biosyntheses of the branched-chain amino acids, isoleucine and valine, using TPP as a cofactor. It was hypothesised that thil encodes a functional AHAS which has a reduced affinity for TPP resulting in a thiamine auxotrophic phenotype. The thil allele was cloned and enzyme assays were carried out which supported this hypothesis. Sequencing analysis and site-directed mutagenesis revealed that the thil phenotype was produced as a result of a single point mutation which caused the conserved amino acid substitution D176E. Hence in this study an amino acid residue potentially important in the binding of TPP to the AHAS enzyme is identified.

Thesis (M.S.) -- University of Maryland, College Park, 2005.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005.
Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

The MAT∝ locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins responsible for determining the ∝cell type. The MAT∝1 gene encodes ∝1, a positive regulator of ∝cell-specific genes, whereas the MAT∝2 gene encodes a negative regulator of a cell-specific genes (∝2). MAT∝2. (in conjunction with the MATα1 gene) also determines the α/∝ diploid cell type by repressing haploid-specific genes. ∝2 exerts its effect at the transcriptional level in the ∝ cell by binding to a sequence located upstream of α cell-specific genes. The present study undertook to examine, through in vitro genetic manipulation, the structure/function relationship of the MAT∝ regulatory proteins, particularly∝2, in their role as gene regulators. The construction of mutant MAT∝2 genes containing termination codons at various points within the gene, and subsequent transformation of the mutant genes into mat∝2 yeast, indicated that the carboxy-terminal one-third of the gene product was necessary for full repressor activity in the haploid as well as in the diploid. A segment within the carboxy-terminal one-third of ∝2 displays some homology to the higher eukaryote homeo domain as well as to a prokaryotic bihelical DNA-binding structural motif. This region of the gene was subjected to semi-random missense mutagenesis in vitro and the mutant genes were analyzed by transformation into strains containing chimaeric genes that encode β-galactosidase from ∝2 and a1/∝2. repressible promoters. In this manner it was demonstrated that most of those residues in ∝2. which correspond to conserved amino acids in the prokaryotic DNA-binding structure and in the homeo domain are essential for the two repressor activities of ∝2. Several mutations more severely affected the ability of ∝2 to repress α-specific genes than haploid-specific genes. Analysis of the temperature dependence of the activities of some of the mutants was consistent with the existence of a helix-turn-helix structure at this region of the protein. Finally, further analysis of some of these mutants in vitro confirmed that the observed defect correlated with a loss of DNA-binding activity.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

The glycine decarboxylase complex (GDC) of Succharomyces cerevisiae composed of four subunits (P, H, T and L) and plays an important role in the interconversion of serine and glycine and balancing the one-carbon unit requirements of the cell. It also enables the cell to use glycine as sole nitrogen source. This study was concerned with characterising the molecular mechanism of transcriptional regulation of the GCVgenes encoding the subunits of the GDC. The important findings of this work can be summarised as follows: i) Transcription of the GCV genes are regulated by glycine and rich nitrogen sources, which are mediated by different cis-acting elements. The LPDl gene did not show a glycine response since its transcriptional regulation is distinct from that of the other genes encoding the GDC subunits. ii) Glycine analogues or serine did not affect expression of GCV2, and therefore glycine probably needs to be metabolised to effect the glycine response of the GCV genes. iii) The repression of the GCV2 gene expression by rich nitrogen sources is mediated by a sequence between -227 and -205 of GCV2, and NCR-regulatory mutant studies showed that repression is not directly controlled by the known NCR system. iv) The glycine response of GCV2 is mediated by a motif (the glycine regulatory region; GRR; 5'-CATCN7CTTCTT-3') with CTTCTT at its core. Additional sequence immediately 5' of this motif (between -310 to -289) plays a minor role for the gene's full glycine response. v) The GRR of the GCV genes can mediate the glycine response by either activation or repression, indicating that the transcription factor(s) mediating the glycine response is/are dual-functional in nature. vi) Studies of GCV2 gene expression using different regulatory mutants showed that expression of the gene is further modulated by other transcription factors such as and Baslp which are distinct from the glycine response and possibly involved in setting up the basal expression level. vii) I n vitro studies of the GRR-protein interaction revealed THF affects the affinity of the DNA-binding protein(s) for the GRR. The importance of THF in regulation of the GCV2 gene was also shown in vivo using a foll mutant that is unable to synthesise any folates. THF or a C1-bound derivative of it acts as a ligand for the transcription factor, thus influencing transcription of the GCV genes in the appropriate physiological manner. viii) Using heparin-Sepharose chromatography fractions, four complex formations (complex I to IV) were observed with the GRR. The protein responsible for one of these was separable from the others. EMSA profiles using the GRR of the GCVI and GCV2 genes (in the presence or absence of THF) were very similar, indicating that these genes bind the same proteins and are regulated in a similar manner. ix) Mutation of the CTTCTT motif within the GRR caused significant reduction in in vitro DNA-protein complex formation, however, THF addition overcame this reduction. x) Only complex II formation was observed with a DNA fragment spanning -322 to -295, and THF affected this complex formation. xi) Footprinting analyses of complex I revealed that the binding protein protected the GRR of the GCV2 gene from DNaseI activity. This protein is an excellent candidate for the glycine response regulatory protein. Titration experiments using EMSA showed that this protein can dimerise. A preliminary genome-wide analysis of the S. cerevisiae transcriptome was carried out using miniarray membrane hybridisation. This investigated the global transcriptional changes within the cell in response to the addition of glycine into the medium. Identification of genes related to various cellular processes including onecarbon metabolism gave an insight into the regulation of the cellular metabolic flow, especially that of one-carbon metabolism. The results indicated that: xii) Glycine is transported into mitochondria to be used as substrate for the GDC which (with mitochondria1 SHMT) produces serine that is subsequently utilised for the various one-carbon metabolic pathways, such as methionine synthesis and purine synthesis. xiii) A gene of unknown function (YER183C) which showed homology to the gene for human 5,lO-CH-THF synthetase was identified from gene-array analysis to be upregulated on glycine addition, indicating the protein encoded by this gene may be involved in balancing the metabolic flow between methionine and purine synthesis when THF pools are disturbed by glycine addition. xiv) Addition of glycine to the medium also triggers the expression of other metabolic genes related to amino acid biosynthetic pathways and that of many other genes which are not directly related to one-carbon metabolism. This may be due to prolonged culturing with glycine in the medium resulting in altered expression of genes mediated by one or more secondary factors. These may reflect an adaptive response rather than a direct consequence of glycine induction. On the basis of the above data, a model for the mechanisms regulating glycine response is presented.

Le tréhalose est un disaccharide non réducteur de glucose généralement considéré chez la levure comme une réserve en hydrates de carbone. Il est largement répandu au sein du règne végétal et constitue donc une source de carbone pour les microorganismes. Nous avons mis en évidence l'existence de deux voies d'assimilation du tréhalose exogène chez Saccharomyces cerevisiae. La voie majeure est dirigée par la tréhalase acide Ath1p dont nous avons montré que l'activité est à 90% localisée dans l'espace périplasmique. Le crible d'une collection de 4500 mutants nous a ensuite permis d'étayer nos connaissances sur le fonctionnement de cette voie. La voie alternative correspond à un couplage entre le transporteur de haute affinité, Agt1p et la tréhalase neutre Nth1p. Nos travaux suggèrent aussi que le produit du gène NTH2, de fonction inconnue, possède une fonction tréhalase neutre. Le profil atypique de consommation du tréhalose en phase stationnaire semble alors être dû à l'activation de cette tréhalase. Enfin, nous avons observé deux phénomènes d'oscillation distincts au cours de nos cultures discontinues, phénomènes qui peuvent se produire de manière concomitante et/ou consécutive, et dont les pérennisations semblent être liées à la mobilisation du tréhalose intracellulaire
Trehalose is a non reducing disaccharide of glucose considered a storage carbohydrate for yeast, as well as glycogen. Trehalose is also widespread in plants and that makes it an alternative carbon source for microorganisms. In this work, we showed that Saccharomyces cerevisiae is able to grow on trehalose using two distinct pathways. Applying the invertase assay (on whole cells) to trehalase, we firstly showed that more than 90% of acid trehalase activity of Ath1p is periplasmic, splitting of the disaccharide into glucose. A throughput screening performed on a 4500-mutant collection then completed our understanding of this major pathway. We also found that the second, independent pathway corresponds to a coupling between the Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p. Nevertheless this pathway is only an alternative route because of several constraints such as the control of AGT1 expression by the MAL system or the inactivation/degradation process of Agt1p exhibited during growth on trehalose. Our results also suggest that the Nth2p protein whose function has not been identified yet displays a neutral trehalase activity. The atypical trehalose consumption profile during stationary phase seems to be related to the Nth2p activation. In batch cultivation, we also observed oscillations, probably favoured by the purely oxidative metabolism exhibited during growth on trehalose. We then described two types of oscillations that can occur simultaneously or consecutively during the culture, and show that these phenomena do not occur in the absence of active neutral trehalase in the cells

Genetic variation is the raw material upon which natural selection acts to spread advantageous phenotypic traits throughout populations. Studies in the yeast Saccharomyces cerevisiae have begun to uncover the extent of genetic variation within yeast populations, but the fitness effects of this variation are still poorly understood. Furthermore, the interplay between genotype, envkonment and fitness only recently begun to be explored at the molecular level. The aun of this thesis is to explore this complex relationship.

DNA interstrand cross-links (ICLs) present a major challenge to cells, covalently binding the two DNA strands together and preventing fundamental cellular processes, such as transcription and replication. Failure to repair such les ions can lead to the formation of DNA double-strand breaks (DSBs), gross-chromosomal rearrangements (GCRs) and ultimately, cell death. ICL-inducing agents have been used clinically for the management of cancers, and increased tolerance of these lesions has been implicated in the development of resistance to such treatments which might, in part, be mediated by DNA repair. This repair requires the orchestration of many different DNA repair mechanisms, such as nucleotide excision repair (NER), trans-lesion synthesis (TLS) and homologous recombination (HR), and the action of factors required specifically for ICL repair, such as the 5'-3' exonuclease Pso2/SNM IA and members of the vertebrate Fanconi anemia (FA) pathway. Disruption of Saccharomyces cerevisiae PS02 confers sensitivity to ICL-inducing agents but no other forms of DNA damage. Here I show that in the absence of Ps02, a complex of the MutSα (Msh2-Msh6) mismatch repair (MMR) factor, the mitochondrial replication and repair protein Mgm101 and the FANCM homologue Mph1 facilitates the loading of Exol to chromatin to allow for the repair of ICLs. Moreover, S. cerevisiae homologues of the FA proteins FANCJ (Chll) and FANCP (Slx4) are also required for this FA-like pathway, which leads to TLS, HR and non-homologous end-joining (NHEJ). The Ps02-dependent and FA-like pathways are controlled by NER and protect cells against DSBs, GCRs and the constitutive activation of the intra S-phase checkpoint following ICL induction. In vertebrate cells, incision of ICLs requires structure-selective endonucleases that cleave stalled replication forks that converge on an ICL. The work presented in this thesis shows that RadI, the S. cerevisiae homologue for one of these endonucleases (XPF), is essential for the repair of ICLs in the absence of NER. Rad I controls an S-phase specific pathway, distinct from the Pso2-dependent and FA-like pathways, but requires TLS and HR. These data suggest that the repair of ICLs in S. cerevisiae and higher eukaryotes is more comparable than previously thought, making budding yeast an attractive model organism for the study of ICL repair.

Dissertation (PhD)--Stellenbosch University, 2007.
PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology.
AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC 3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek of dit op te neem en intrasellulêr te benut. Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/ transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen 0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te fermenteer. Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1 ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium. Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende anaerobe geassosieer word. Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut. Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg word.

Thesis (PhD)--University of Stellenbosch, 1999.
ENGLISH ABSTRACT: Hemicellulose, consisting mainly of xylan, ranks after cellulose, as the most abundant group of renewable polysaccharides in agricultural biomass. Xylan is a complex polymer consisting of a β D 1,4 linked xylopyranoside backbone, which may contain substituents. Enzymatic hydrolysis of xylan involves the action of a number of different hydrolytic enzymes. The yeast Saccharomyces cerevisiae has been used extensively in traditional food and beverage processes (baking, brewing and winemaking), as well as for the production of ethanol (potable alcohol and fuel extenders) and single-cell protein (protein supplements in food and animal feed). S. cerevisiae therefore has complete GRAS (Generally Regarded as Safe) status. However, the yeast S. cerevisiae can neither degrade nor utilize complex polysaccharides, including xylan. Through recombinant DNA technology, S. cerevisiae can be complemented by heterologous polysaccharase-encoding genes, thereby broadening its substrate range and facilitating a direct bioconversion of polysaccharides to valuable commodities, such as potable ethanol, protein supplements and industrial enzymes. In this study, the successful expression and co-expression of a β xylanase gene (Trichoderma reesei xyn2) and two β xylosidase genes (Bacillus pumilus xynB and A. niger xlnD) in S. cerevisiae, is described. Expression of these genes was obtained with the aid of multi-copy episomal yeast plasmids pJC1, pDLG1, pDLG4 and pRLR1. These plasmids contain either the derepressible alcohol dehydrogenase 2 (ADH2) or the constitutive phosphoglycerate kinase 1 (PGK1) promoter and terminator sequences. The enhanced production of recombinant enzymes by S. cerevisiae in a rich medium, without the risk of losing the episomal vector, was obtained by disrupting the uracil phosphoribosyltransferase (FUR1) gene in the plasmid-containing S. cerevisiae strains. This step ensured auto-selection of the URA3-bearing expression plasmids in rich growth medium. High level expression of the T. reesei β xylanase gene in S. cerevisiae enabled the yeast to degrade xylan to short xylo-oligosaccharides, but very little monomeric D xylose was formed. Both β xylosidase genes enabled S. cerevisiae to degrade short xylo-oligosaccharides like xylobiose and xylotriose. Co-expression of the β xylanase and the B. pumilus β xylosidase led to a small increase in the β xylanase activity, but a substantial decrease in the amount of β xylosidase activity. This recombinant yeast strain was unable to degrade xylan to D xylose. Expression of the T. reesei β xylanase with the A. niger β xylosidase gene enabled this strain to completely degrade xylan to its monomeric constituents, D xylose.
AFRIKAANSE OPSOMMING: Hemisellulose, wat hoofsaaklik uit xilaan bestaan, is ná sellulose, die volopste hernubare polisakkaried in landbouafval. Xilaan is 'n komplekse polimeer wat bestaan uit 'n β-D-1,4-gekoppelde xilopiranoseruggraat wat in sommige gevalle ook sykettings bevat. Ensimatiese afbraak van xilaan benodig die werking van hele aantal hidrolitiese ensieme. Die gis Saccharomyces cerevisiae word al vir baie jare in die voedsel- en drankbedryf (bak van brood en die maak van bier en wyn), asook vir die produksie van etanol (vir menslik gebruik en as brandstof aanvuller) en enkelselproteïene (proteïenaanvulling vir mens en dier) gebruik en het daarom volledige GRAS (Generally Regarded As Safe) status. Ongelukkig kan S. cerevisiae nie komplekse polisakkariede, xilaan ingesluit, afbreek of as koolstofbron benut nie. Met behulp van rekombinante-DNA-tegnologie kan S. cerevisiae gekomplementeer word met die nodige gene wat kodeer vir polisakkariedafbrekende ensieme om sodoende die gis in staat te stel om 'n wyer verskeidenheid van substrate af te breek en te benut. Dit sal lei tot die direkte bio-omskakeling van polisakkariede na bruikbare produkte soos etanol, proteïenaanvullers en ensieme vir industriële gebruik. In hierdie proefskrif word die suksesvolle uitdrukking asook die gesamentlike uitdrukking van 'n xilanasegeen (Trichoderma reesei xyn2) en twee β-xilosidasegene (Bacillus pumilus xynB en A. niger xlnD) in S. cerevisiae beskryf. Multikopie episomale plasmiede pJC1, pDLG1, pDLG4 en pRLR1 met die glukose onderdrukbare alkoholdehidrogenase 2 (ADH2) of die konstitutiewe fosfogliseraatkinase 1 (PGK1)- promoter en -termineerder is vir hierdie doel gebruik. Verhoogde produksie van die rekombinante ensieme deur S. cerevisiae in 'n ryk medium, sonder dat die gis die episomale plasmiedvektore verloor is moontlik gemaak deur die urasielfosforibosieltransferasegeen (FUR1) van hierdie giste te onderbreek met behulp van die LEU2-geen. Op hierdie manier word daar outomaties vir giste wat die URA3-uitdrukkingsplasmiede bevat geselekteer, selfs in ryk medium. Hoë vlak uitdrukking van T. reesei se xilanasegeen het S. cerevisiae in staat gestel om xilaan tot kort xilo-oligosakkariede af te breek, maar byna geen monomeriese D-xilose is gevorm nie. Albei die β-xilosidasegene het die gis in staat gestel om kort xilo-oligosakkariede soos xilobiose en xilotriose na D-xilose af te breek. Die gesamentlike uitdrukking van die xilanasegeen en B. pumilus se β-xilosidase geen het 'n klein toename in die xilanase-aktiwiteit tot gevolg gehad, maar 'n drastiese afname in die β-xilosidase-aktiwiteit. Hierdie rekombinante ras kon dus nie xilaan tot xilose afbreek nie. Uitdrukking van T. reesei se β-xilanasegeen saam met die β-xilosidasegeen van A. niger, het S. cerevisiae in staat gestel om xilaan tot sy monomeriese boustene, D-xilose, af te breek.

Precise and complete replication of the genome is essential for a cell. Chromosome replication follows a defined temporal order, depending on the efficiency and timing of the replication origins. However, the mechanism regulating origin activity has not been properly explained to date. Yeast replication origins are very well characterized and well studied. Genome wide replication in yeast was detailed through deep sequencing in various studies. In yeast, there are multiple replication origins for the complete replication of the genome. In Saccharomyces cerevisiae, there are ~400 replication origins, which are also referred to as Autonomously Replicating Sequences. Replication origins have varied levels of activity and varying times of activation. There are many lines of evidences, which suggest that the origins function differently in mitotic and meiotic cell cycles. It was thought that same origins function both during mitosis and meiosis. However, there is a difference in the replication timings of both the cell cycles, the reason for which is not known. Meiotic cell cycle is longer than the mitotic cell cycle. By using the plasmid-based assays, specific origins were selected and origin activity was analyzed during mitosis and meiosis to see if individual origins show any differences in origin activity. For all the origins tested, the meiotic activity was found to be less than the mitotic activity, which provides a possible explanation for a longer pre-meiotic S phase. Most of the confirmed yeast replication origins are present in the intergenic regions of the chromosome. Due to the presence of majority of replication origins in the intergenic regions and not on the genes, it was thought that the gene transcription might be detrimental to origin activity, hence not supporting the existence of an origin on a gene. Careful analysis of genome wide replication data along with plasmid based ARS assays confirmed that a few replication origins are present within genes. Assays were preformed to study the relation between transcription and origin activity both during mitosis and meiosis. Mitotic origin activity was shown to have no known affect from gene transcription. However, due to some unknown technical faults or other reasons, assays to find out transcription and meiotic activity were not successful.

Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological appearance in response to a variety of extracellular and intracellular signals. The processes involved in morphogenesis are well characterised in this organism, but the exact mechanism by which information emanating from the environment is integrated into the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly understood. Considerable progress has, however, been made. The processes are investigated on various levels including: (i) the nature of the signals required to elicit a morphological adaptation, (ii) the mechanism by which these signals are perceived and transmitted to the nucleus for gene transcription regulation (signal transduction pathways), (iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the relationship between cell cycle regulators and factors required for alterations in cellular shape. The focus of this study was on elements involved in the regulation of one of these morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal differentiation normal oval yeast cells become elongated and mother and daughter cells stay attached after cytokinesis to give rise to filaments. These filaments are able to penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin remodelling is a prerequisite for the formation of elongated cells during pseudohyphal development and invasive growth. Its main contribution to this event is the directing of vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth at the cell periphery. In order to fulfil this cellular function, actin is regulated on several levels. Signal transduction pathways that are activated in response to external nutritional signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal differentiation. For this reason a literature review was compiled to introduce various aspects of actin-structure, the regulation of this structure and the functions actin performs during morphogenesis. The connection between signal transduction elements involved in morphological processes and actin remodelling is also reviewed. This study entailed the genetic analysis of numerous factors involved in the regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several transcriptional regulators playing a role in these phenomena were investigated. Apart from the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p, additional elements ranging from transporters to G-proteins, were also investigated. Mutant strains deleted for one or more of these factors were constructed and tested to assess their abilities to form filaments that penetrate the growth substrate, and to utilise starch as a carbon source. Complex genetic relationships were observed for various combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act independently in controlling invasive growth and starch metabolism, suggesting that these factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p functions at the confluence of several signalling pathways controlling the transcriptional regulation of genes required for invasive growth and starch degradation. Different nutritional signals were also found to differentially regulate specific signalling elements during the invasive growth response. For example, Tec1 p requires Msn1 p activity in response to growth on media containing a limited nitrogen source. This dependency, however, was absent when invasive growth was tested on glucose and starch media. Evidence was also obtained that confirmed the transcriptional co-regulation of MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for starch degradation. Unpublished results indicated that several transcriptional regulators of invasive growth also exert an effect on starch metabolism. The data generated during this study complemented and confirmed published results. It also contributed to the compilation of a more detailed model, integrating the numerous factors involved in these signalling processes.
AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae gisselle beskik oor die vermoë om hul morfologiese voorkoms in responstot 'n verskeidenheid van ekstrasellulêre en intrasellulêre seine te verander. Die prosesse betrokke by morfogenese is goed gekarakteriseerd in hierdie organisme, maar die presiese meganisme waardeur inligting vanuit die omgewing geïntegreer word in die reguleringvan die aktien-sitoskelet en die gisselsiklus, word nog nie ten volle verstaan nie. Aansienlike vordering in die verband is egter gemaak. Die prosesse word op verskeie vlakke ondersoek, insluitende: (i) die aard van die seine wat benodig word om 'n morfologiese aanpassing te inisïeer; (ii) die meganisme waardeur hierdie seine waargeneem en herlei word na die selkern vir die regulering van geen-transkripsie (seintransduksie paaie); (iii) die rol van die sitoskelet, spesifiek aktien, in morfogenese en (iv) die verhouding tussen selsiklusreguleerders en faktore wat benodig word vir verandering in selvorm. Hierdie navorsing fokus op elemente betrokke by die regulering van een van hierdie morfologiese prosesse in S. cerevisiae, naamlik pseudohife-vorming. Gedurende pseudohife-differensiëring neem tipiese ovaalvormige selle 'n verlengde voorkoms aan wat tot die vorming van filamente lei. Hierdie filamente is in staat om die groeisubstraat te penetreer, 'n verskynsel bekend as penetrasie-groei. Aktienherrangskikking is 'n voorvereiste vir die vorming van verlengde selle tydens pseudohife-ontwikkeling. Die hoofbydrae van aktien tot hierdie verskynsel is die oriëntering van uitskeidingsvesikels, wat selwandkomponente en ensieme bevat, na spesifieke areas van selwandgroei op die seloppervlak. Aktien word op verskeie vlakke gereguleer om hierdie sellulêre funksie te vervul. Seintransduksiepaaie wat geaktiveer word in respons tot ekstrasellulêre voedingsseine speel 'n belangrike rol in die regulering van die aktien-sitoskelet tydens pseudohife-differensiëring. Op grond hiervan is 'n literatuuroorsig saamgestel vir die bekendstelling van verskeie aspekte van aktienstruktuur, die regulering van hierdie strukture en die funksies wat deur aktien gedurende morfogenese vervul word. Die verband tussen seintransduksie-elemente betrokke by morfologiese prosesse en aktien herrangskikkingword ook behandel. Hierdie studie het die genetiese analisering van verskeie faktore betrokke by pseudohife-differensiëring, penetrasie-groei en styselmetabolisme, behels. Verskeie transkripsionele reguleerders wat In rol speel in hierdie prosesse was bestudeer. Buiten die transkripsiefaktore Mss11p, Msn1p, Ste12p, F108p,Phd1P en Tec1p, was addisionele faktore, wat gewissel het van transporters tot G-proteïene, ook ondersoek. Mutante-rasse met geendelesies vir een of meer van hierdie faktore is gekonstrueer en getoets om vas te stel hoe dit hul vermoë raak om penetrerende filamente te vorm, asook om te bepaal of stysel as koolstofbron gebruik kan word. Komplekse genetiese interaksies vir verskeie kombinasies van hierdie faktore is waargeneem. Dit was waargeneem dat F108p,Msn1p en Ste12p onafhanklik funksioneer tydens die regulering van penetrasie-groei en styselmetabolisme, wat impliseer dat hierdie faktore deur verskillende seintransduksiepaaie gereguleer word. Mss11 p word beskou as In onmisbare rolspeler in hierdie prosesse en dit kom voor asof hierdie protein as 'n stroom-af faktor is en vereis word vir die funksionering van Msn1p, F108p, Ste12p en Tec1p. Phd1p is egter 'n uitsondering, aangesien veelvuldige kopieë van PHD1 die effek van 'n MSS11-delesie gedeeltelik oorkom. Die data impliseer dat Mss11 p by die samevloei van verskeie seintransduksiepaaie, benodig vir die transkripsionele regulering van gene betrokke by penetrasie-groei en styselmetabolisme, funksioneer. Dit was ook waargeneem dat verskillende voedingsseine die faktore betrokke by die penetrasie-groeirespons differensieel reguleer. Tec1 p byvoorbeeld benodig Msn1paktiwitieit in respons tot groei op media met 'n beperkte stikstofbron. Hierdie afhanklike interaksie is egter afwesig wanneer penetrasie-groei bestudeer word op glukose- en styselmedia. Resultate wat die gesamentlike transkripsionele regulering van MUC1 en STA2 bevestig, is ook verkry. MUC1 kodeer vir 'n mukienagtige proteïen wat benodig word vir pseudohife-vorming en penetrasie-groei, terwyl STA2 kodeer vir 'n glukoamilase essensieël vir styselafbraak. Ongepubliseerde resultate dui daarop dat verskeie transkripsionele reguleerders van penetrasie-groei ook In effek uitoefen op styselmetabolisme. Die data wat gegenereer is tydens hierdie studie komplementeer en bevestig reeds gepubliseerde resultate. Dit het ook bygedra tot die samestelling van 'n gedetaileerde model wat die verskillende faktore, betrokke by hierdie seintransduksieprosesse, integreer.

El mètode tradicional utilitzat en la producció de vins escumosos (com el cava i el champagne) es caracteritza per una segona fermentació que té lloc dins l’ampolla. Aquesta segona fermentació presenta unes característiques molt específiques com són un alt contingut d’etanol, una pressió de CO2 creixent, baixa disponibilitat de nutrients i baixa temperatura. En aquesta tesi, s’ha analitzat en primer lloc l’efecte de factors ambientals sobre la cinètica fermentativa de Saccharomyces cerevisiae durant la segona fermentació, mitjançant el seguiment de la segona fermentació amb afròmetres. En segon lloc hem analitzat quin efecte tenen pràctiques habituals com l’addició de nutrients al vi base sobre la composició final del vi escumós, mitjançant l’anàlisi per HPLC del contingut d’aminoàcids i polisacàrids del vi i la seva capacitat escumant (mosalux). Finalment ens vàrem proposar identificar les bases genètiques de la segona fermentació mitjançant la detecció i validació de Quantitative Trait Locus (QTL). Els resultats obtinguts ens han permès identificar la temperatura, el vi base utilitzat, la soca de llevat i la font de nitrogen utilitzada en l’aclimatació del llevat com els factors que tenen més impacte en la segona fermentació. En segon lloc respecte a la composició final del vi escumós, hem pogut detectar que l’adició de nitrogen al vi base afavoreix, l’alliberament d’aminoàcids. Mentre que, l’adició de llevats secs inactius, promou la alliberació de polisacàrids i afavoreix l’escumabilitat del vi escumós. Finalment, hem identificat quatre gens, la variació al·lèlica dels quals explica la variació fenotípica observada entre soques.
El método tradicional utilizado en la producción de vinos espumosos (como el cava y el champagne) se caracteriza por una segunda fermentación que tiene lugar dentro de la botella. Esta segunda fermentación presenta unas características muy específicas como son un alto contenido de etanol, una presión de CO2 creciente, baja disponibilidad de nutrientes y baja temperatura. En esta tesis, se han analizado en primer lugar el efecto de factores ambientales sobre el desarrollo de la segunda fermentación, mediante su seguimiento con afrómetros. En segundo lugar hemos analizado qué efecto tienen prácticas habituales como la adición de nutrientes al vino base sobre la composición final del vino espumoso, mediante el análisis por HPLC del contenido de aminoácidos y polisacáridos del vino y su capacidad espumante (mosalux). Finalmente nos propusimos identificar las bases genéticas de la segunda fermentación mediante la detección y validación de “Quantitative Trait Locus” (QTL). Los resultados obtenidos nos han permitido identificar la temperatura, el vino base utilizado, la cepa de levadura y la fuente de nitrógeno utilizada en la aclimatación de la levadura como los factores que tienen mayor impacto en la segunda fermentación. En segundo lugar respecto a la composición final del vino espumoso, hemos podido detectar que la adición de nitrógeno al vino base favorece, la liberación de aminoácidos. Mientras que, la adición de levaduras secas inactivas, promueve la liberación de polisacáridos y favorece la espumabilidad del vino espumoso. Por último, hemos identificado cuatro genes, la variación alélica de los que explica la variación fenotípica observada entre cepas.
The traditional method used to produce sparkling wines (such as cava and champagne) is characterized by a second fermentation that takes place inside the bottle. This second fermentation has very specific characteristics such as a high ethanol content, increasing CO2 pressure, low temperature and low nutrient availability. In this thesis, we have firstly analyzed the effect of environmental factors on fermentation kinetics of Saccharomyces cerevisiae during the second fermentation, by monitoring the second fermentation development using aphrometers. Secondly, we analyzed what effect have common practices such as adding nutrients to the base wine on the final composition of the sparkling wine by HPLC analysis of content of amino acids and polysaccharides and its foaming capacity (mosalux) of the sparkling wine. Finally we aimed to identify the genetic basis of the second fermentation using Quantitative Trait Locus (QTL) mapping and validation approach. The results obtained enabled us to identify the temperature, the base wine used, the yeast strain and source of nitrogen used in the acclimatization of yeast as the factors that have the highest impact in the second fermentation kinetics. Secondly, with respect to the final composition of sparkling wine, we have found that the addition of nitrogen to the wine base favors the release of amino acids. While the addition of inactive dry yeast, promotes the release of polysaccharides and favors the foaming propoerties of the sparkling wine. Finally, we have identified four genes whose allelic variation explains the phenotypic variation observed among strains.

Previous studies have shown that mitochondrial petites of the yeast Saccharomyces cerevisiae appear to have some advantage over their wild type counterparts in terms of fermentative performance. In this thesis evidence is presented that inhibition of the respiratory chain by chemical or genetic means leads to an increase in ethanol formed per unit sugar and a decrease in biomass per unit sugar. The genetic studies involve a mutation in formation of subunit III of the cytochrome £ oxidase complex (pet!22) and a mutation in putative mitochondrial pyruvate transport (tpyl). The chemical study involves inhibition of mitochondrial pyruvate transport by a specific inhibitor alpha-cyano-4-hydroxycinnamic acid. To try and discover more of the nature of the tpyl mutation a gene was cloned from a yeast centromeric genomic library which complemented the mutation. The insert containing the complementing gene was reduced in size and sequenced. An open reading frame containing 1698 nucleotides coding for 566 amino acids was discovered. After comparison to databases the gene appeared to be undiscovered to date. No similarity was found between the translated protein and others on databases with the exception of the FAS2 gene of Penicillium patulum. Studies of isolated mitochondria and cell growth pattern, of the tpyl mutant, revealed data typical of a classical petite mutation rather than a mutation in a regulatory gene as described by the original paper describing the mutant.

The cell division of Saccharomyces cerevisiae is controlled by the action of pheromones at the G1 phase of the cell cycle. A general method was developed for the isolation of constitutive mutants in the pheromone response pathway. Recessive alleles of the SCG1 gene (encoding the α subunit of a G protein) were isolated as well as a dominant mutation in the STE4 gene (encoding the β subunit of a G protein). Analysis of double mutants suggested that the STE4 gene product functions after the SCG1 product but before the STE5 gene product. Double mutants carrying either scg1 or STE4Hp1 constitutive alleles together with the temperature-sensitive unresponsive mutation, ste5-3ts, showed arrest and recovery when shifted from 34° C to 22° C. Recovery from the constitutive signal was independent of the receptor. The STE4Hp1 sst2 ste5ts triple mutant was not able to recover from arrest, suggesting that an SST2-dependent mechanism is involved in recovery of the STE4Hp1 mutant from constitutive arrest. In contrast, the scg1-7 sst2 ste5ts triple mutant recovered only partially suggesting that even though SST2 gene product is probably involved in recovery of the scg1-7 mutant, this mutant can recover by an SST2-independent mechanism. This implies existence of another, SST2-independent postreceptor recovery mechanism. The scg1-null mutant do not recover from constitutive arrest (J. Hirschman, personal communication). Both recovery mechanisms probably operate at the G protein step. Isolation of a constitutive allele of STE5 allowed the definition of its site of action as being after the STE4-controlled step. In addition, constitutive activation of the pheromone pathway by STE5Hp1 mutation was found to be partially dependent on the STE4 and STE18 gene products, the β and γ subunits of a G protein. A comprehensive genetic model is presented to explain the mechanisms of signal transduction and recovery.

L'intégrité des génomes est constamment compromise par l'agression d'agents externes et internes. Plusieurs mécanismes assurent son maintien, parmi lesquels sont la recombinaison homologue (HR), la réparation post-réplicationnelle (PRR) ou la jonction non-homologue. Il existe plusieurs processus pour décider du type de réparation à utiliser et assurer la fiabilité de la réparation. Chez la levure Saccharomyces cerevisiae, l'un d'entre eux repose sur Phélicase Srs2, qui est au carrefour de processus du métabolisme de l'ADN, notamment pendant la réplication ou elle participe à la décision entre HR et PRR. Nous avons étudié les fonctions du gène SRS2 par un crible de dosage synthétique létal (SDL) à grande échelle. Nous avons surexprimé SRS2 ainsi que deux mutants de sa fonction hélicase dans la collection de plus de 4800 souches délétés pour des gènes non essentiels et avons identifié 274 gènes dont la fonction est requise pour la viabilité des cellules en conditions de surexpression. Ces nouvelles interactions concernent des gènes impliqués dans diverses fonctions cellulaires : métabolisme de l'ADN, métabolisme de l'ARN ou encore trafic vésiculaire. Nous nous sommes intéressés au métabolisme de l'ADN et avons décrit qu'un excès de Srs2 est toxique lors de la réplication de l'ADN, indépendamment de son activité hélicase, du fait de son interaction avec le PCNA. Cet effet active le checkpoint de réplication qui dépend de la protéine Mrcl. Par ailleurs, Srs2 est phosphorylée et sumoylée en réponse à sa surexpression. Nous avons identifié 20 sites de phosphorylation et 4 sites de sumoylation qui sont possiblement liées à la tolérance cellulaire de la protéine surexprimée
The genome of all living organisms is constantly compromised by both exogenous and endogenous damaging agents. Multiple mechanisms maintain genome integrity, which include homologous recombination (HR), post-replication repair (PRR) and non-homologous end joining. Many processes are involved in making the decision between repair mechanisms and ensuring the quality of the repair. In Saccharomyces cerevisiae, one of them relies in specialized proteins like the Srs2 helicase. This protein is at the crossroads of DNA metabolic processes, notably during DNA replication at the decision point between HR and PRR. We investigated the functions of the SRS2 gene in Saccharomyces cerevisiae by means of an original high throughput synthetic dosage lethality screen (SDL). We overexpressed SRS2, as well as two helicase-dead mutants, in the collection of over 4800 mutants deleted for non-essential genes and have identified 274 genes that are required for cell viability upon overexpression. These new interactions concern genes involved in various cellular functions: DNA metabolism, RNA metabolism and vesicular trafficking. W€ first focused our study on DNA metabolic functions and describe that an increased dosage of Srs2 is toxic during DNA replication. The toxicity is independent of the helicase function of the protein, relies on the interaction of Srs2 with the PCNA replication clamp and activates the DNA replication checkpoint, which depends on the Mrcl. Moreover, Srs2 is phosphorylated and sumoylated in response to its overexpression. We identified 4 sumoylation sites and 20 phosphorylation sites on Srs2 that may be responsible for tolérance of the cell to the overexpressed protein

La méthode traditionnelle utilisée pour produire des vins mousseux (comme cava et champagne) est caractérisée par une seconde fermentation qui a lieu à l'intérieur de la bouteille. Cette deuxième fermentation présente des caractéristiques très spécifiques telles qu’une teneur élevée en éthanol, la pression croissante de CO2, une basse température et une faible disponibilité des nutriments. Dans cette thèse, on a tout d'abord analysé l'effet des facteurs environnementaux sur la cinétique de fermentation de Saccharomyces cerevisiae au cours de la seconde fermentation. Deuxièmement, on a analysé l'effet de pratiques communes, telles que l'ajout de nutriments au vin de base, sur la composition finale du vin mousseux. Enfin, nous avons cherché à identifier la base génétique de la variabilité phénotypique observée pendant la seconde fermentation en utilisant une approche Quantitative Trait Locus (QTL). Les résultats obtenus ont permis de déterminer la température, le vin de base utilisé, la souche de levure et la source d'azote utilisée dans l'acclimatation de levure comme les facteurs qui ont la plus grande incidence dans la cinétique de la seconde fermentation. Deuxièmement, par rapport à la composition finale du vin mousseux, nous avons trouvé que l'addition d'azote dans le vin base favorise la libération des acides aminés. Bien que l'ajout de levure sèche inactive favorise la libération des polysaccharides et les propriétés moussantes du vin mousseux. Enfin, on a pu identifier quatre gènes dont la variation allélique explique la variation phénotypique observée parmi les souches
The traditional method used to produce sparkling wines (such as cava and champagne) is characterized by a second fermentation that takes place inside the bottle. This second fermentation has very specific characteristics such as a high ethanol content, increasing CO2 pressure, low temperature and low nutrient availability. In this thesis, we have firstly analyzed the effect of environmental factors on fermentation kinetics of Saccharomyces cerevisiae during the second fermentation, by monitoring the second fermentation development using aphrometers. Secondly, we analyzed what is the effect of common practices such as adding nutrients to the base wine on the final composition of the sparkling wine by HPLC analysis of content of amino acids and polysaccharides and its foaming capacity (mosalux) of the sparkling wine. Finally we aimed to identify the genetic basis of the second fermentation using Quantitative Trait Locus (QTL) mapping and validation approach. The results obtained enabled us to identify the temperature, the base wine used, the yeast strain and source of nitrogen used in the acclimatization of yeast as the factors that have the highest impact in the second fermentation kinetics. Secondly, with respect to the final composition of sparkling wine, we have found that the addition of nitrogen to the wine base favors the release of amino acids. While the addition of inactive dry yeast, promotes the release of polysaccharides and favors the foaming properties of the sparkling wine. Finally, could identify four genes whose allelic variation explains the phenotypic variation observed among strains

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are usually used for the production of whisky. One of the first steps in the production of whisky is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is performed to disrupt and solubilise the starch granules to make them more accessible for enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and then saccharified by glucoamylase and a debranching enzyme. Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in existing industrial fermentations because of their low ethanol tolerance, slow growth rate, catabolite repression, poorly characterised genetics and lack of GRAS (Generally Regarded As Safe) status. This study is divided into two sections. The aim of the first section was to clone a gene (LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid, YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and terminator (PGKh) expression cassette. This recombinant plasmid was designated pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid assays revealed that the recombinant yeast secreted active a-amylase into the medium. The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C. The aim of the second part of the study was to construct recombinant strains of S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase (SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids expressing the individual coding sequences under the control of the PGK1 p-PGK1.,- expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively; two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were transformed into a laboratory strain of S. cere visiae , L1278b. By homologous recombination, each plasmid was integrated into the yeast genome at the ura3 locus. S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of a-amylase activity when assayed for amylolytic activity in a liquid medium. This recombinant strain resulted in the most efficient starch utilisation in batch fermentations, consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation. The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette gave the highest levels of glucoamylase activity.' These results confirmed that co-expression of a-amylase and/or glucoamylase synergistically enhance starch degradation. This study paves the way for the development of efficient starch-degrading strains of S. cerevisiae for the production of whisky, beer and biofuel ethanol.
AFRIKAANSE OPSOMMING: Styselbevattende landbougewasse kom wydverspreid voor as die substraat vir die produksie van brandstofetanol, drinkbare spiritualië of bier, enkelselproteïen en hoëfruktose graanstroop. Styselbevattende gewasse, soos mielies, rog, gars en koring, word gewoonlik vir die produksie van whisky gebruik. Die eerste stap in die produksie van whisky is om die stysel by temperature bo 1DOOG te kook. Hierdie jelatinisasie stap word uitgevoer om die styselkorrels te versteur en vloeibaar te maak sodat hulle meer toeganklik vir ensimatiese hidrolise is. Na dié kookproses word die stysel deur o-arnilases vervloei en dan deur glukoamilases en 'n vertakkingsensiem versuiker. Lipomyces kononenkoae en Saccharomycopsis filuligera skei hoogs effektiewe a-amilases en glukoamilases uit, wat dit twee van die effektiefste rou-stysel-afbrekende giste bekend, maak. L. kononenkoae en S. fibuligera kan egter nie in reeds bestaande industriële fermentasies gebruik word nie, as gevolg van hulle lae etanoltoleransie, stadige groeitempo, katabolietonderdrukking, swak gekarakteriseerde genetika en gebrek aan ABAV (Algemeen Beskou As Veilig) status. Hierdie tesis is in twee afdelings verdeel. Die doel van die eerste deel was om 'n geen (LKA2) wat vir 'n nuwe, unieke styselafbrekende ensiem kodeer, te kloneer, 'n tweede a-amilase (Lka2p) van L. kononenkoae. LKA2 is in 'n multikopie plasmied, die gis episomale plasmied, YEp352, onder beheer van die fosfogliseraatkinasepromotor- en termineerder-kasset (PGK1 p-PGK1 r), gekloneer. Hierdie rekornbinante plasmied is pJUL3 genoem en in 'n laboratoriumras van Saccharomyces cerevisiae, L:1278b, getransformeer. Plaat- en vloeibare-ensiem toetse het getoon dat die rekombinante gis aktiewe a-amilase in die medium uitskei. Die optimum pH vir Lka2p is 3.5, is en die optimum temperatuur 60oG. Die doel van die tweede deel van die studie was om rekombinante rasse van S. cerevisiae te konstrueer wat a-amilases en/of glukoamilases uitskei. Die individuele gene is toe in 'n gis-integreringsplasmied, Ylp5, onder beheer van die PGK1p-PGK1,ekspressiekasset, gekloneer. Twee inheemse giste is op grond van hulle vermoë om stysel te benut geselekteer, L. kononenkoae en S. filuIigera, as geen donors. Agt konstrukte bevattende die L. kononenkoae se a-amilasegene, LKA 1 en LKA2, en S. filuligera se a-amilasegeen (SFA 1) en glukoamilasegeen (SFG1), moes gekonstrueer word: vier _enkel-kasset plasmiede wat die individuele koderende sekwense onder beheer van die PGK1 p-PGK1, ekspressiekasset uitdruk, wat onderskeidelik plPLKA 1, pIPLKA2, plPSFA 1 en plPSFG1 lewer; twee dubbel-kasset plasmiede (wat beide LKA 1 en LKA2 onder beheer van die PGK1 p-PGK1,ekspressiekasset, en SFA 1 en SFG1 met hulle onderskeie inheemse promotors en termineerders) uitdruk, wat onderskeidelik pIPLKA1/2 en plPSFAG lewer, en twee enkel-kasset plasmiede wat SFA 1 and SFG1 met hulonderskeie inheemse promotors en termineerders, en wat onderskeidelik pSFA 1 en pSFG1 lewer. Die onderskeie konstrukte is in 'n laboratoriumras van S. cerevisiae, L1278b, getransformeer. Deur middel van homoloë rekombinasie, is die onderskeie plasmiede in die ura3-lokus van die gisgenoom geïntegreer. S. cerevisiae L1278b, getransformeer met plPLKA 1/2, LKA 1 en LKA2 onder die beheer van die PGK1 ~PGK1 ïekspressiekasset, het die hoogste vlakke van a-amilase aktiwiteit gelewer toe dit vir amilolitiese aktiwiteit in vloeibare medium getoets is. Hierdie rekombinante ras het stysel die effektiefste benut, nl. 80% van die stysel en 'n opbrengs van 6 gIL etanol na 156 ure in lotfermentasies. Die ras wat SFG1 onder beheer van die PGK1~PGK1ïekspressiekasset uitdruk, het die hoogste vlakke van glukoamilase-aktiwiteit gelewer. Hierdie resultate bevestig dat die gesamentlike uitdrukking van a-amilase- en/of glukoamilase-ensieme styselafbreking sinergisties . bevorder. Hierdie studie baan die weg vir die ontwikkeling van 'n effektiewe styselfermenterende ras van S. cerevisiae wat moontlik gebruik kan word vir die produksie van whisky en biobrandstofalkohol.

Thesis (MSc)--Stellenbosch University, 2002.
Full text to be digitised and attached to bibliographic record.
ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer.
AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.

CP1 is a sequence specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved centromere DNA element I (CDEI) of yeast centromeres. The gene encoding CP1, which was designated CEP1 for centromere protein 1, was cloned and sequenced. CEP1 encodes a highly acidic protein of molecular weight 39,400. CEP1 was mapped to a position 4.6 centiMorgans centromere distal to SUP4 on the right arm of chromosome X. Phenotypic analysis of cep1 mutants demonstrated that yeast strains lacking CP1 are viable but have a 35% increase in cell doubling time, a ninefold increase in the rate of mitotic chromosome loss, and are methionine auxotrophs. Detailed analysis of the mitotic chromosome-loss phenotype showed that the loss is primarily due to chromosome nondisjunction (2:0 segregation). During meiosis cep1 null mutants exhibited aberrant segregation of centromere containing plasmids, chromosome fragments, and chromosomes. The predominant missegregation event observed was precocious sister segregation. The mutants also displayed a nonrandom 20% decrease in spore viability. Missegregation of chromosomes accounted for some but not all of this decreased spore viability, the remainder of which is presumed to be related to the pleiotropic consequences of the cep1 mutation. Together with the observed mitotic missegregation phenotype the results are interpreted as suggesting that CP1 promotes sister chromatid-kinetochore adhesion. The following conclusions are based on my mutational analysis of CP1: (1) CP1 is normally present in functional excess, (2) the C-terminal 143 amino acids are sufficient for full CP1 function in chromosome segregation and methionine metabolism, and (3) while DNA binding is apparently necessary for function, DNA binding per se is not sufficient. All of the mutations which caused an observable phenotype affected both centromere function and methionine metabolism. In addition, a direct correlation was observed in the degree to which both phenotypes were affected by different mutations. None of the mutant proteins displayed trans-dominant effects in a wild type background; however, two nonfunctional DNA binding-competent mutants exerted a dominant negative effect on the ability of PHO4 to suppress cep1 methionine auxotrophy. The data are consistent with a model in which CP1 performs a similar function at centromeres and promoters.

SGS1, a Saccharomyces cerevisiae 3 '-5' DNA helicase, is a homologue of the Escherichia coli RecQ gene. It is essential for genomic stability of both during mitosis and meiosis. The purpose of this thesis is to provide a better understanding of the role of this helicase during meiotic recombination. In meiosis, SGS1 mutant cells display a decrease in sporulation efficiency and spore viability. In addition, the unusual spore viability pattern observed in SGS1 mutants cannot be explained solely by meiosis I or meiosis II missegregations. These problems could be partially explained by defects in mitotic chromosome segregation or problems with meiotic S-phase. Cytological experiments demonstrating an increase in synapsis initiation complexes and axial associations in sgs1Delta could be explained by an early function of Sgs1p in meiosis, such as the unwinding of inappropriate strand invasion events. Consistent with this, we observe increased gene conversion, increased homeologous recombination and increased interaction between sister choromatids.;Recent observations have suggested that, Sgs1p and Top3p in S. cerevisiae, and the human orthologue protein BLM, in conjunction with the Top3alpha protein, can dissolve double Holliday junctions. Physical analyses of double-strand break repair in meiosis, combined the genetic analysis of this work, indicate a late function of the Sgs1 protein in the dissolution of double Holliday junctions. We have shown an unusual class of tetrads in which non-sister spores and recombinant spores are dead. We interpret this as a consequence of the failure to untangle intertwined chromatids. This defect in SGS1 mutant strains could be explained by either the presence of pre-meiotic S-phase catenates, a defect in crossover resolution and/or a defect in the dissolution of closely spaced double Holliday junctions.

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

The genome of an organism is not uniformly mutagenic. The overall aim of this project was to identify cis and trans factors that may contribute to such differential mutagenic activities within the genome using Saccharomyces cerevisiae. A well-characterised recombination reporter construct, hisG-URA3-hisG, was separately introduced into five different locations of chromosome III. Each locus had differing features with respect to their replication dynamics: three replication termination sites, two of which coincided with “Replication Slow Zones” (RSZ; Cha and Kleckner, 2002), one replication origin and a region of no discernable feature. Fluctuation analysis was used to assess the rate of URA3 inactivation at each locus. First, the effects of temperature, a replication inhibitor hydroxyurea, and ploidy were assessed. Significant differences in mutation rates existed in diploid strains heterozygous for the construct in these conditions, but not in respective haploids. The effects of inactivating various genes known to be involved in genome stability were also examined. Elimination of an essential signal transduction protein, Mec1p, or a DNA helicase required for efficient replication, Rrm3p, led to an increase in mutation rates only in diploid strains. No statistically significant effect was seen when a top2 temperature-sensitive allele was used in either haploids or diploids. In general, no cis effect was observed in any of these mutant backgrounds. The nature of genetic alterations associated with URA3 inactivation was also determined by Southern analysis for the five loci. The analysis revealed that the nature of genetic alteration is regulated in a cis manner, as URA3 inactivation was either exclusively via recombination or by small changes depending on the location of the reporter construct. These findings reveal some unexpected ways in which cis and trans factors may regulate mutagenic events in budding yeast. These will be discussed in context of eukaryotic genome instability in general.

Huntington’s disease (HD) is a fatal late onset neurodegenerative disorder that is caused by an expansion of a polyglutamine encoding tract in the huntingtin gene. This expansion in mutant huntingtin (mHtt) causes the protein to misfold, aggregate and leads to widespread cellular dysfunction principally through a toxic gain of function mechanism. The expression of mHtt in baker’s yeast recapitulates many of the cellular phenotypes observed in mammalian models. Here I have exploited a yeast HD model to perform genetic screens for mammalian cDNAs whose overexpression suppresses mHtt toxicity. We identified 102 protective genes, which are significantly enriched for components of the translational machinery. Further investigations revealed that translational efficiency but not fidelity is decreased in yeast expressing mHtt. Interestingly, overexpression of a subset of these genetic modifiers further decreases the efficiency of translation in yeast. I have validated a subset of the translation related suppressors in a neuronal model of HD and observed that their overexpression significantly reduces mHtt-dependent caspase activation. In yeast mHtt aggregation and toxicity are dependent on the presence of the Rnq1 prion. Previous genetic screens in our laboratory have identified a number of other putative yeast prions as modifiers of mHtt toxicity. I have further investigated their ability to alter mHtt aggregation and to substitute for the Rnq1 prion. I have observed that overexpression of the aggregation prone Ybr016w and the Sup35 prion partially restores aggregation of mHtt in a dose-dependent manner in a rnq1Δ strain. My studies have revealed new information about the role of genetic modifiers in mHtt toxicity and aggregation, and may help inform novel therapeutic strategies for HD.

The aim of this work was to establish the relative contribution of genetic regulation of the PYK1, PFK1 and PFK2 genes to the control of glycolysis. A series of isogenic mutant strains were constructed where the promoters and 5' untranslated sequences of the PYK1, PFK1 and PFK2 genes were replaced with those from PGK1. In addition , a second series of mutant strains were constructed where synthesis of Pyk1p and Pflkp was driven by the PGK1_Δuas promoter. These latter series of mutants were designed to contain weak expression of Pyklp and Pflkp. Analysis of UKC1 (PGK::PYK1) in shake flask cultures revealed similar growth rates on glucose and on lactate and similar rates of ethanol production and glucose consumption to those of the wild-type strain. This suggested that the native genetic regulation did not appear to play a significant role in the control of glycolysis. Nonetheless, analysis of this strain in the fermentor revealed that genetic regulation of PYK1 may be important in co-ordinating Pyk1p synthesis, under the conditions studied. Analysis of YKC11 (PGK_Δuas::PYK1) in both shake flask and fermentor experiments showed that genetic control was important in maintaining Pyk1p levels in order to sustain glycolytic flux. Shake flask analysis of the single and double PFK mutants under the control of the PGK1 promoter revealed that the genetic regulation of the PFK1 and PFK2 genes did not appear to be important in the control of glycolysis. Weak expression of the PFK1 and PFK2 genes, under the control of the PGK_Δuas promoter showed the importance of genetic regulation in maintaining Pflkp levels to support glycolytic flux, under the conditions studied.

Mestrado em Biologia Molecular e Celular
Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.
O código genético é geralmente visto como imutável, no entanto várias alterações à sua forma padrão são conhecidas. Uma das mais notáveis acontece em várias espécies do género Candida, onde o codão Leu-CUG é descodificado como serina por um novo RNA transferência (Ser-tRNACAG). O laboratório de acolhimento fez um grande progresso ao reverter a alteração atípica do código genético do fungo patogénico humano C. albicans, usando uma combinação de tRNAs mutantes, recombinação genética e evolução forçada. Estes resultados levantaram a hipótese que as ambiguidades sintéticas do codão, combinadas com evolução experimental, poderem libertar os codões do seu estado fixo. Nesta tese testamos esta hipótese usando S. cerevisiae como modelo biológico. Geramos ambiguidade em codões específicos, de forma bifásica, envolvendo a deleção de genes de tRNA, seguida pela expressão de tRNAs não-cognatos capazes de compensar o tRNA eliminado. Tendo como base a ideia que os codões raros são mais suscetíveis a alterações que aqueles usados frequentemente, usamos duas estirpes knock-out, nas quais não existem os tRNAs cognatos capazes de descodificar os codões raros CUC-Leu e AGG-Arg. Exploramos então a vulnerabilidade destes codões pela construção de tRNAs mutantes que incorporam erradamente Ser nestes locais. Estas estirpes recombinantes foram evoluídas ao longo do tempo, usando evolução experimental. Apesar de ter havido um forte impacto negativo na taxa de crescimento de estirpes que expressam o tRNA mutante a altos níveis, esta expressão a baixos níveis teve pouco impacto no fitness celular. Descobrimos que não só a ambiguidade do codão, mas também destabilizações da pool de tRNAs endógenos têm um impacto negativo na taxa de crescimento. Após a evolução, as estirpes com elevada expressão do tRNA mutante recuperaram significativamente em vários parâmetros de crescimento, o que mostra que estas adaptam-se e exibem maior tolerância à ambiguidade do codão. Através do sistema repórter fluorescente desenvolvido monitorizamos a incorporação errónea de Ser, o que nos indica que a Ser está de facto a ser incorporada e que, possivelmente, a alteração da identidade do codão foi atingida. Apesar das consequências negativas gerais da ambiguidade do codão, demonstramos que os codões capazes de tolerar a perda do seu tRNA cognato, conseguem também tolerar a incorporação elevada de Ser. Isto levanta a hipótese que estes codões podem ser recodificados para outros aminoácidos naturais e/ou artificiais para a produção de proteínas com novas propriedades, contribuindo assim para o campo da Biologia Sintética e Biotecnologia.