Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by browse.metadata.advisor "Divol, Benoit"

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    Characterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains
    (Stellenbosch : Stellenbosch University, 2010-03) Malherbe, Daniel Francois; Van Rensburg, P.; Pretorius, I. S.; Du Toit, M.; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology, 2010.
    ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production.
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    Investigating the lipid requirements of wine-related non-Saccharomyces yeast
    (Stellenbosch : Stellenbosch University, 2022-03) Mbuyane, Lethiwe Lynett; Divol, Benoit; Bauer, Florian; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Lipids are structural components of the yeast plasma membrane which is a barrier between the intra- and extracellular environment. The lipid bilayer also contains proteins responsible for transport, signalling and cell function. Changes in the lipid composition are necessary for yeast to adapt to unfavourable conditions that occur during alcoholic fermentation. Numerous studies focused on the lipid metabolism of Saccharomyces cerevisiae exist and previous reports have shown that ergosterol, oleic and palmitoleic acids enhance fermentation performance, ethanol stress resistance and impact aroma production. On the other hand, little is known about non- Saccharomyces yeasts. Instead, previous studies showed that selected non-Saccharomyces yeasts produce lipids different from S. cerevisiae but the impact of these differences on lipid metabolism and fermentation performance was not evaluated. The aim of this study was to evaluate the impact of exogenous lipids on fermentation kinetics, metabolite production and ethanol stress resistance in wine relevant non-Saccharomyces yeasts. Protocols focused on fatty acid and sterol extraction are tedious, requiring large sample and solvent volumes. Furthermore, the analytical methods focused on detecting sterols and fatty acids on one instrument are limited. Lipid extraction and analysis via Gas Chromatography – Mass Spectrometry was therefore optimized in yeast cells and synthetic media. The data showed that while only monounsaturated fatty acids (oleic and palmitoleic acids) were detected in S. cerevisiae cells, the non-Saccharomyces yeasts in this study accumulated linoleic and linolenic acids in addition to monounsaturated fatty acids. The data in this study showed that Metschnikowia pulcherrima, Kluyveromyces marxianus and Torulaspora delbrueckii are sensitive to the presence of lipids in the extracellular environment. Indeed, the impact of fatty acid and sterol containing mixtures on fermentation performance was species-specific. This species-specific response to the different lipid mixtures could be linked to acetyl-CoA availability and redox balance regulation based on the differences in alpha- ketoglutarate, acetate, malate and succinate biosynthesis during fermentation. K. marxianus and M. pulcherrima were found sensitive to ethanol shock with the latter being the most sensitive. The addition of unsaturated fatty acids did not drastically increase ethanol tolerance. Instead, K. marxianus and M. pulcherrima were sensitive to ethanol in media supplemented with polyunsaturated fatty acids indicating that when these compounds (which impact membrane fluidity) are taken up into the cell, they do not counteract the inhibitory effects of ethanol in these strains. Furthermore, the presence of unsaturated fatty acids increased lipid droplet accumulation following ethanol shock, possibly in an effort to reduce the toxic effects of these compounds on the cell during ethanol stress. Overall, this study showed that M. pulcherrima, K. marxianus as well as T. delbrueckii require specific lipid combinations for an increase in fermentation rate. Furthermore, the species-specific response to lipid mixtures may be due to differences in the lipid composition, acetyl-CoA metabolism and redox balance. Moreover, this study showed that the presence of polyunsaturated fatty acids during ethanol shock may be detrimental for selected non- Saccharomyces yeasts. Specific lipid mixtures and oxygen sparging could be suggested to winemakers interested in increasing non-Saccharomyces yeast persistence during alcoholic fermentation and ultimately the metabolic footprint as well as quality of wine.
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    Investigation into physical and metabolic interactions within the wine yeast ecosystem
    (Stellenbosch : Stellenbosch University, 2021-12) Luyt, Natasha Alethea; Bauer, Florian; Divol, Benoit; Setati, Mathabatha Evodia ; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: The microbial community of the wine ecosystem consist of filamentous fungi, bacteria and yeast. These organisms interact and compete for space and nutrients throughout fermentation. Since yeast are the primary contributors to alcoholic fermentation, various studies have described and characterized the biotic and abiotic factors which may influence yeast-yeast interactions. Through this search for a fundamental understanding of interactions, physical and metabolic interaction have emerged as pivotal drivers of population dynamics during fermentation. Nevertheless, these interactions remain elusive and the molecular mechanisms behind them remain poorly described. This study aimed at characterizing cell-cell and metabolic interactions between Saccharomyces cerevisiae and Lachancea thermotolerans from a phenotypic and molecular viewpoint. To achieve these outcomes, synthetic grape must fermentations were performed in a compartmentalised bioreactor, followed by a transcriptomic analysis which evaluated the effect of cell-cell and metabolic contact on gene expression and finally, a qRT-PCR approach, further evaluating the expression of specific genes of interest. The data confirmed the existence of an antagonistic relationship between S. cerevisiae and L. thermotolerans, which has been previously reported. It was observed that the presence of S. cerevisiae caused cellular death in L. thermotolerans in a cell-cell and metabolic contact dependant manner and the former appears more important in S. cerevisiae’s strategy to outcompete L. thermotolerans. In turn, the data also suggest that the metabolic activity of L. thermotolerans has a negative effect on the culturability of S. cerevisiae. Analysing the transcriptomic responses as a result of cell-cell and metabolic contact revealed distinct responses in both yeasts. S. cerevisiae reacted in a targeted manner, reinforcing its cell wall through the up-regulation of genes associated with maintaining cell wall integrity and structural components of the cell wall. L. thermotolerans showed a different response, with in particular strongly up-regulated heat shock genes, some of which have previously been linked to interspecies interaction. Both yeasts avoided co-aggregation by expressing adhesion genes less when in physical contact. Genes of interest were identified and their expression was further monitored throughout different stages of fermentation and investigated as to whether these responses were generic or species-specific. In S. cerevisiae, PAU, TIR2, HSP12 and FLO gene regulation occurred in a species-specific manner when evaluated in co-fermentations. While the regulation of adhesion FLO genes occurred in a species-specific manner between two closely related non-Saccharomyces yeasts, the role of HSP genes appeared to be conserved between the two. The avoidance of co-adhesion appeared to be a generic response, both in S. cerevisiae and non-Saccharomyces yeasts. The data provide novel insights into the transcriptomic responses to cell-cell contact in S. cerevisiae and non-Saccharomyces yeasts. Furthermore, the data provides a basis for future annotation of the S. cerevisiae genome to include the role of genes in ecological interactions.
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    Molecular screening of lactic acid bacteria enzymes and their regulation under oenological conditions
    (Stellenbosch : University of Stellenbosch, 2011-03) Mtshali, Phillip Senzo; Du Toit, Maret; Divol, Benoit; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions.
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    Transcriptional regulation of the endo-polygalacturonase-encoding gene in Saccharomyces cerevisiae
    (Stellenbosch : University of Stellenbosch, 2010-03) Louw, Campbell Trout; Van Rensburg, P.; Divol, Benoit; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities.