Doctoral Degrees (Institute for Wine Biotechnology)

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Browsing Doctoral Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Bauer, Florian"

Now showing 1 - 5 of 5
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    Carnitine metabolism and biosynthesis in the yeast Saccharomyces cerevisiae
    (Stellenbosch : University of Stellenbosch, 2009-12) Franken, Jaco; Bauer, Florian; Strauss, Erick; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications.
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    Defining the chemical features of wine perception
    (Stellenbosch : Stellenbosch University, 2018-03) Fairbairn, Samantha; Bauer, Florian; Da Silva Ferreira, A. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: All wines evoke a product recognition, regardless of quality and cultivar, but what is the origin of this feature? The prevalence of this wine concept suggests that its formation occurs independent of the varietal, and ageing-related aromas, and is therefore potentially a function of yeast metabolism. Yeast utilise the nutrients present in grape must to produce biomass, and metabolites which ultimately signify the conversion of grape juice to wine. Consequently, the nutrient composition is highly influential on the aromatic outcomes of alcoholic fermentation. Synthetic grape must is widely used to evaluate all facets of the fermentation process but there remains much to learn. In this study, the impact of two nutrients, namely, amino acids and anaerobic factors, were evaluated with regard to their impact on yeast growth and aroma production under fermentative conditions. This work also examines the extent to which yeast de novo metabolism, both primary and secondary metabolism, contributes to the formation of the wine-like feature. In a single amino acid context, a linear relationship was apparent between the amino acid concentration and the production of their associated volatile products. This relationship was evaluated in more complex amino acid mixtures and as expected, this linear relationship was lost. Nonetheless, a significant degree of responsiveness between the amino acid and its catabolites remained. The impact of sterol (plant or yeast derived) or unsaturated fatty acid treatments, individually, as well as in combinations, were compared for their contributions to biomass formation and aroma production. Sterols had a greater impact on biomass development, as the fermentations treated with only unsaturated fatty acids displayed a poorer response. Moreover, they differently impacted aroma production. The unsaturated fatty acid lowered the production of acetate esters, medium chain fatty acids and their esters, whereas sterol supplementation generally bolstered the production of all compounds measured. This work highlights the importance of anaerobic factor management during winemaking. Although these nutrients certainly impact wine aroma, this study also sought to examine the degree to which these nutrients contribute to wine (product) recognition. Using a novel fermentation-based approach, Saccharomyces cerevisiae converted a synthetic grape must into a wine-like product. These synthetic products underwent sensory evaluations to rate the product’s resemblance to wine as well as to describe the aroma. This sensory data was used as a decision-making tool to decide upon treatments to be studied in subsequent fermentations. Ultimately, a wine-like character was created by altering the anaerobic factor composition of a synthetic grape must. The use of this synthetic grape must would allow for the more meaningful sensory characterisation of these synthetic products, in addition to providing a wine-like matrix used to evaluate the sensory implications of wine odorants.
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    Evaluating the effect of oxygen addition on yeast physiology, population dynamics and wine chemical signature in controlled mixed starter fermentations
    (Stellenbosch : Stellenbosch University, 2017-12) Sekhawat, Kirti; Setati, Mathabatha Evodia ; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH SUMMARY: The use of commercial starter cultures of non-Saccharomyces yeast, usually together with Saccharomyces cerevisiae, has become a trend in the global wine industry in the past decade. Depending on the specific species of non-Saccharomyces yeast, the procedure may aim at enhancing aroma and flavour complexity of the wine, reduce acetic acid levels, and/or lower the ethanol yield. However, the contribution of non-Saccharomyces yeast strains depends on several factors, and in particular on the strains ability to establish significant biomass and to persist for a sufficient period of time in the fermentation ecosystem. For an effective use of these yeasts, it is therefore important to understand the environmental factors that modulate the population dynamics of such environments. In this study, we evaluated the effect of oxygen addition on yeast physiology, population dynamics and wine chemical signature in controlled mixed starter fermentations. The population dynamic in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species namely, Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, revealed that oxygen availability strongly influences the population dynamics and chemical profile of wine. However, results showed clear species-dependent differences. Further, experiments were confirmed in Chardonnay Grape juice, inoculated with L. thermotolerans and S. cerevisiae with different oxygen regimes. The results showed a trend similar to those obtained in synthetic grape juice, with a positive effect of oxygen on the relative performance of L. thermotolerans. The results in this study also indicates that continuous stirring supports the growth of L. thermotolerans. We further analysed the transcriptomic signature of L. thermotolerans and S. cerevisiae in single and mixed species fermentations in aerobic and anaerobic conditions. The data suggest the nature of the metabolic interactions between the yeast species, and suggests that specific stress factors are more prominent in mixed fermentations. Both yeasts showed higher transcript levels of genes whose expression is likely linked to the competition for certain metabolites (copper, sulfur and thiamine), and for genes involved in cell wall integrity. Moreover, the transcriptomic data also aligned with exo-metabolomic data of mixed fermentation by showing higher transcripts for genes involved in the formation of aroma compounds found in increased concentration in the final wine. Furthermore, the comparative transcriptomics analysis of the response of the yeasts to oxygen provides some insights into differences of the physiology of L. thermotolerans and S. cerevisiae. A limited proteomic data set aligned well with the transcriptomic data and in particular confirmed a higher abundance of proteins involved in central carbon metabolism and stress conditions in mixed fermentation. Overall, the results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and its impact on yeasts physiology. The transcriptomics data clearly showed metabolic interaction between both yeasts in such ecosystem and provide novel insights into the adaptive responses of L. thermotolerans and S. cerevisiae to oxygen availability and to the presence of the other species.
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    Exploring multispecies interactions between wine-associated yeasts
    (Stellenbosch : Stellenbosch University, 2021-12) Conacher, Cleo Gertrud; Bauer, Florian; Rossouw, Debra; Blassoples-Naidoo, Rene; Stellenbosch University. Faculty of AgriSciences. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The fermentation of grape must to wine is catalysed by a diverse microbial community. Yeast are primary drivers of the associated alcoholic fermentation process and have therefore garnered considerable research interest. The diversity of yeast species present during wine fermentation influences the chemical composition and related sensory properties of wine as a result of the metabolic functioning of particular yeast species in response to abiotic and biotic factors. The latter is a relatively new research field, given that microbiological science has a significant monoculture bias, and as such, there is much still to be understood about the role and mechanisms of biotic stress in wine yeast ecosystems. Moreover, while the wine yeast ecosystem was the model used in this study, there are several other yeast ecosystems of biotechnological importance, including in biofuels production, bioremediation and other food and beverage industries, that would benefit from insight into these biotic stress mechanisms. The current basis of our understanding of the molecular mechanisms of yeast interactions in the wine ecosystem is based on two-species pairings, which keeps the system interaction network uncomplicated. However, there are many more role-players in natural ecosystems, and they do not interact in a linear fashion. At the micro- and macroscopic level, the importance of these often overlooked higher-order interactions has been highlighted in other ecosystems. There is very little information on higher-order interactions in the yeast ecology field, and this must be remedied for predictive understanding of these systems. Here, we sought to address the current status quo in multispecies yeast research, by aiming to develop new tools to investigate the mechanistic basis of interaction in systems comprised of more than two species. Furthermore, the study aimed to generate a greater depth of understanding of these systems, by investigating transcriptional responses of Saccharomyces cerevisiae to co-culture in mixed-species cultures of increasing complexity. Firstly, these aims were achieved by developing a fluorescence-based multi-colour flow cytometric method for tracking of a consortium consisting of wine-associated yeast species. This involved optimizing the genetic modification of the selected environmentally isolated yeast species, followed by extensive validation to confirm the representativeness of the system as well as development of the flow cytometric protocol. This was followed by addressing the pertinent issue of reproducibility in multispecies cultures, and showing the role of the physiological state of pre-cultures in determining their growth performance in three-species and four-species consortia. Finally, to contribute to our understanding of the molecular mechanisms of interaction in non-linear yeast systems, we showed that Saccharomyces cerevisiae expresses a combination of known pair-wise as well as unique genes when grown in a three-species system. By using interactive network visualizations of the generated transcriptomic data, we were able to functionally characterize the cellular responses in more detail than has been done before in similar studies.
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    Organic acid metabolism in Saccharomyces cerevisiae : genetic and metabolic regulation
    (Stellenbosch : Stellenbosch University, 2016-03) Chidi, Boredi Silas; Bauer, Florian; Rossouw, D.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Organic acids are major contributors to the organoleptic properties of wine. Each acid indeed contributes to the overall acidity of the product, which is an essential feature of wine quality. In addition, and an aspect that has been neglected in many evaluations in the past, each acid also imparts its own sensory characteristic to the wine. Changes in organic acid profiles therefore define relevant sensory features of wine beyond the general perception of acidity. The main objective of this study was to investigate how different yeast strains and a number of environmental factors (such as aeration, initial pH, temperature and sugar content) influence the organic acid levels in fermenting musts at three critical physiological stages (exponential, early stationary and late stationary phase). Five commercial wine yeast strains (VIN13, EC1118, BM45, 285 and DV10) were selected and these strains were subjected to two widely differing fermentation conditions. The data showed significant variation in organic acid concentrations in the final product depending on the yeast strain, and a more multifactorial experimental design was adopted to investigate the impact of environmental parameters. The impact on both grape-derived (tartaric, citric and malic acid) and fermentation-derived (succinic, acetic and pyruvic acid) acids was evaluated. Condition-dependent shifts in the production of specific organic acids were observed. The multifactorial experimental design evaluated environmental parameters that can be at least partially controlled or managed in the cellar. The influence of individual and /or combinatorial factors such as temperature, pH and sugar content of the must were also shown to affect acid profiles of the synthetic wines. A further goal of this project was to identify genes that are involved in organic acid metabolism. Transcriptome data of the five yeast strains was analyzed in order to identify genes which showed differential expression between strains and/or time points paralleled by differences in organic acids for the same comparisons. A correlation model was constructed for genes identified in this manner and model predictions were compared/aligned to observed changes in acid levels in response to deletion of the target genes. This approach provided some predictive capacity for modelling the impact of target genes on acid levels. Although some predictions based on gene expression to acid correlations were not validated experimentally, the analysis as a whole provided new insights into organic acid evolution mechanisms of different strains at different stages of fermentation. Overall, the use of a multifactorial experimental design in the current study confirmed existing knowledge and sheds new light on factors which, either on their own or in combination with other factors, impact on individual organic acids in wine. As a practical outcome, the data can serve for the development of guidelines for winemakers with regard to strain selection and management of fermentation parameters in order to better control wine acidity and wine organic acid profiles.