Doctoral Degrees (Institute for Wine Biotechnology)

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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.
Characterisation, evaluation and use of non-Saccharomyces yeast strains isolated from vineyards and must
(Stellenbosch : Stellenbosch University, 2004-03) Jolly, N. P. (Neil Paul); Pretorius, I. S.; Augustyn, O. P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in which yeasts play the most important role regarding the alcoholic (primary) fermentation. These wine-associated yeasts can be divided into Saccharomyces and non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae, which then completes the fermentation. This is especially evident in spontaneously fermenting must, which has a low initial S. cerevisiae concentration. Some non- Saccharomyces yeasts can also be found throughout the fermentation. The non- Saccharomyces presence in the fermentation can affect wine quality, either positively or negatively. A positive contribution could be especially useful to improve wines produced from grape varieties with a neutral flavour profile due to non-optimal climatic conditions and/or soil types. As part of a comprehensive South African research programme, the specific objectives of this study were: the isolation of indigenous non-Saccharomyces yeasts from vineyards and musts; the identification of these isolates; the characterisation and evaluation of predominant species under winemaking conditions; and the development of a protocol for their use in enhancing wine quality. Initially, 720 isolates representing 24 different species, were isolated from grape (vineyard) and must samples taken over three vintages from four distinctly different wine producing regions. The isolates were characterised and grouped utilising biochemical profiles and DNA karyotyping, whereupon representative isolates were identified. The yeast species that had the highest incidence of predominance in the vineyard was Kloeckera apiculafa. However, some vineyard samples were characterised by low numbers or absence of this yeast, which is not according to generally accepted norms. Other species that also predominated in a few of the vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans, Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern linking species to climatic zone was observed. Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and Kloeckera apiculata, were found to predominate in grape must samples. Representative strains consequently received further attention during laboratory and small-scale winemaking trials. A protocol was developed whereby individual species could be used in co-inoculated fermentations with S. cerevisiae in the small-scale production of wine. An improvement in wine quality was achieved and it was found that there was a link between specific species and grape cultivar. The ability of C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results over three vintages showed that the wine produced by the co-inoculated fermentation was superior to that of a reference wine (produced by S. cerevisiae only). The improvement in wine quality was not linked to increased ester content nor were the standard chemical analyses adversely affected. The effects of pH and wine production parameters i.e. 802, fermentation temperature and use of di-ammonium phosphate (DAP), on this yeast followed the same pattern as that known for S. cerevisiae. This study was successfully completed and the developed protocol can be used for the improvement of Chenin blanc wine where additional aroma and quality is needed.
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.
Effect of non-Saccharomyces yeasts and lactic acid bacteria interactions on wine flavour.
(Stellenbosch : Stellenbosch University, 2018-03) Du Plessis, Heinrich Wilbur; Jolly, Neil; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Wine aroma and flavour are important indicators of quality and are primarily determined by the secondary metabolites of the grape, by the yeast that conducts the primary fermentation and also the lactic acid bacteria (LAB) that performs malolactic fermentation (MLF). This is a complex environment and each microorganism affects the other during the wine production process. Therefore, the overall aim of this study was to investigate the interactions between Saccharomyces, non-Saccharomyces yeasts and LAB, and the effect these interactions had on MLF and wine flavour. Contour-clamped homogeneous electric field gel electrophoreses (CHEF) and matrix-assisted laser desorption ionization using time-of flight mass spectrometry (MALDI-TOF MS) were useful tools for identifying and typing of Hanseniaspora uvarum, Lachancea thermotolerans, Candida zemplinina (synonym: Starmerella bacillaris) and Torulaspora delbrueckii strains. Hanseniaspora uvarum strains had β-glucosidase activity and Metschnikowia pulcherrima strains had β-glucosidase and protease activity. Only Schizosaccharomyces pombe and C. zemplinina strains showed mentionable malic acid degradation. Candida stellata, C. zemplinina, H. uvarum, M. pulcherrima and Sc. pombe strains were slow to medium fermenters, whereas L. thermotolerans and T. delbrueckii strains were found to be medium to strong fermenters, comparable to S. cerevisiae. The effect of non-Saccharomyces yeast species on MLF varied and inhibition was found to be strain dependent. In a Shiraz winemaking trial where seven non-Saccharomyces strains were evaluated in combination with S. cerevisiae and three MLF strategies, the C. zemplinina and the one L. thermotolerans isolate slightly inhibited LAB growth in wines where yeast and LAB were inoculated simultaneously. However, the same effect was not observed during sequential inoculation of LAB. Mixed culture fermentations using non-Saccharomyces yeasts contained lower alcohol levels, and were more conducive to MLF than wines produced with S. cerevisiae only. Yeast treatment and MLF strategy resulted in wines with significantly different flavour and sensory profiles. Yeast selection and MLF strategy had a significant effect on berry aroma, but MLF strategy also had a significant effect on acid balance and astringency of wines. In a follow up trial, H. uvarum was used in combination with two S. cerevisiae strains, two LAB (Lactobacillus plantarum and Oenococcus oeni) species and three MLF strategies. One of the S. cerevisiae strains had an inhibitory effect on LAB growth, while H. uvarum in combination with this S. cerevisiae strain had a stimulatory effect on MLF. Simultaneous MLF completed faster than sequential MLF and wines differed with regard to their chemical and sensory characteristics. Isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, 2-phenyl acetate, isobutanol, 3-methyl-1-pentanol, hexanoic acid and octanoic acid were important compounds in discriminating between the different wines. Yeast treatment had a significant effect on fresh vegetative and spicy aroma, as well as body and astringency of the wines. The LAB strain and MLF strategy had a significant effect on berry, fruity, sweet associated and spicy aroma, as well as acidity and body of the wines. Mid-infrared (MIR) spectroscopy was used to differentiate between wines produced with the selected Saccharomyces and non-Saccharomyces yeast combinations, LAB species and MLF strategies. This study provides valuable information about the interactions between non-Saccharomyces, Saccharomyces yeast, LAB and MLF strategies, and how important pairing of strains are to ensure successful AF and MLF. Furthermore, the results also showed how these interactions can be applied to diversify wine flavour.
Espacement studies on unirrigated grafted Pinot noir (Vitis vinifera L.)
(Stellenbosch : Stellenbosch University, 1990) Archer, Eben; Goussard, P. G.; Stellenbosch University. Faculty of AgriSciences. Department of Viticulture and Oenology and Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The effect of vine spacing on root distribution, plant and soil water status, some physiological aspects as well as vegetative and reproductive growth of Vitis vinifera L. cv. Pinot noir, grafted onto 99 Richter (Vitis Berandieri var. Las Sorres x Vitis rupestris var. du Lot) was investigated. Vine spacings used were 1,0 m x 0 .5 m, 1,0 m x 1,0 m, 2,0 m x 1,0 m, 2,0 m x 2,0 m, 3,0 m x 1,5 m, and 3,0 m x 3,0 m. This trial was conducted on a medium potential soil without irrigation in the Stellenbosch district. Root density was directly affected by vine spacing and it increased and decreased with closer and wider spacing respectively. The larger contact surface between roots and soil in the case of closer spaced vines, increased the utilization of soil water and nutrients. This increased depletion of soil water, induced water stress earlier in the growth season with the result of a timely arrestment of shoot growth. This phenomenon brought about advantageous characteristious in canopy density and canopy microclimate. Vine spacing also affected cordon length per vine as well as total cordon length per hectare, thus affecting the distribution and orientation of the canopy. Closely spaced vines induced shorter cordons per vine, but the total cordon length per hectare was higher than that of widely spaced vines. Although a smaller crop was produced per vine in the case of closer spacings, the total yield per hectare was higher than in the case of wider spewed vines. In addition, a better potential for bunch nutrition was ensured by a higher leaf surface : fruit mass ratio in the case of closer spaced vines. The low canopy densities of closer spaced vines, together with a better water-supply early in the season (bud break to pea size) ensured a higher physiological activity than in the case of wider spaced vines. As the season progressed, however, this situation was reversed so that the shoot elongation of closer spaced vines ceased before that of wider spaced vines. This phenomenon was advantageous for bunch nutrition, affecting both grape composition and wine quality. The subterranean and above-ground changes brought about by closer vine spacing augmented both yield and quality. A better wine quality was obtained through a higher sugar concentration, lower acid concentration and better colour of the grapes. A higher yield per hectare stemmed from an increase in total cordon length per hectare. These results were realized for a medium potential soil without irrigation. More luxurious conditions will probably induce more vegetative growth, necessitating wider in-row vine spacing for the best yield and quality. The ideal vine spacing for a given locality is dictated by soil potential, rootstock and scion cultivar and cultivation practices such as irrigation and fertilization. It is probably wrong to standardise vine spacing for all vineyards on a specific farm.
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.
Exploring consumers’ risk perception in wine retail decision-making : insights for Chenin blanc
(Stellenbosch : Stellenbosch University, 2019-04) Van der Colff, Nadia; Nieuwoudt, Helene; Pentz, Chris; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: South Africa is a large wine producing country without a wine-drinking culture. Therefore, the development of the domestic wine market is a priority of the Wine Industry Strategic Exercise (WISE). The study of risk perception is known to provide insights, valuable for marketing initiatives aiming to gain market share. Previous researchers identified significant perceived risks and risk-reducing strategies for wine as a generic category. However, this study was a pioneering effort to explore and describe consumer risk perception of a specific wine varietal. Chenin blanc, South Africa’s most planted wine grape variety, accounts for 23% of all varieties crushed for wine production. However, from 2011-2017, domestic Chenin blanc sales appeared to be low in the South African 750ml bottled white wine category. Therefore, the aim of this study was to explore and describe consumers’ perceived risk of Chenin blanc wine. As South African consumers purchase wine primarily from retail stores, the study focused exclusively on consumers’ retrospective, in-store decision-making of white wine. An exploratory sequential mixed methods research approach was followed. Using semi-structured, personal interviews, consumers of white wine (n=8) were firstly interviewed to gain an understanding of Chenin blanc perceptions. Qualitative data, in combination with theory, were used to develop a measurement instrument, utilised in the sequential core quantitative phase. The measurement instrument was pilot tested (n=62) to assess internal reliability. Consequently, an online survey was used to collect data from a sample (n=2051) of wine consumers. Statistical analysis confirmed that the scales used were valid and reliable. Being the first instrument to measure wine varietal-specific perceived risk, further recommendations were made to improve some construct items. The results show that there were significant differences in consumers’ quality perception, subjective knowledge, purchase frequency and perceived goodness-of-fit for occasions between white wine varietals. Based on a comparison between Chenin blanc and the white wine category in general, Chenin blanc was perceived to be less available with a lower amount of information available, while respondents indicated to be more risk averse and lacking self-confidence to evaluate Chenin blanc in a purchase situation. The main perceived risks in the case of Chenin blanc were functional, time and financial risks, while respondents indicated to use another wine varietal as a risk-reducing strategy. Age appeared to have little influence on respondents’ Chenin blanc perceived risk, but there were significant differences between ethnic groups’ Chenin blanc perceived risk. Potential target markets for Chenin blanc were identified with strategies recommended to reduce Chenin blanc perceived risk. Results of this study make a valuable contribution, not only to the South African wine industry, but also to the international body of knowledge on consumers’ wine decision-making. The exploratory sequential mixed methods research approach and varietal-specific measurement instrument can be replicated to study other struggling varietals or even regions-of-origin aiming to gain market share. The target market identified, with strategies to reduce Chenin blanc-specific perceived risks, can be used by the South African wine industry to develop a Chenin blanc marketing plan, aiming to increase market share.
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.
Functional analysis of candidate terpenoid biosynthetic genes isolated from grapevine
(Stellenbosch : Stellenbosch University, 2021-03) Bezuidenhout, Ilse-Marie; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Terpenoids are a group of compounds found in various organisms, with diverse functions, and can be broadly grouped into primary or specialised (secondary) metabolites. This compound diversity is achieved from the universal precursors for terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), through the activity of various enzymes. Isoprenyl diphosphate synthases (IDSs) incorporate the IPP and DMAPP precursors into compounds of varying lengths that can then serve as the substrate for terpene synthases (TPSs) to produce terpenes. Each of these steps incorporates the opportunity to further increase terpene diversity. In addition, terpenes can be modified into other products through the activity of various enzymes to increase the product range even further. Terpenoids play an important role in Vitis vinifera (grapevine), not only in the normal functioning of the plant, but also in more specialised roles involving plant–environment interactions. In terms of their economic value, terpenes, such as mono- and sesquiterpenes, contribute to wine aroma – which has supported the study of these compounds and how these metabolites are biosynthesised. Grapevine genome annotation has shown a moderately sized IDS gene family and a TPS gene family that has undergone extensive expansion. However, many of these genes have not been functionally characterised. Putative annotation based on sequences is not always accurate or does not provide a realistic indication of gene function. Functional characterisation of IDS and TPS genes involves heterologous expression followed by analysis of the produced compounds. In this study, various heterologous systems were used and evaluated with the aim of characterising grapevine genes that putatively encode IDS and TPS enzymes. In this study, the novel annotation of a eucalyptol synthase and the re-annotation of a previously characterised gene to α-thujene synthase was achieved. Various expression systems were assessed, with Saccharomyces cerevisiae deemed a viable alternative to the typical use of Escherichia coli. Different plant systems were also investigated, with Nicotiana benthamiana found to be a good option in terms of ease of use and results obtained. An in-depth study of geranylgeranyl diphosphate synthase (VviGGPS) was also conducted using an established transgenic tobacco population. The population was characterised in terms of morphology, physiology, and metabolites. The possible role of gibberellin was also determined based on the results of these analyses.
Genetic investigation and characterization of killer toxins secreted by non-Saccharomyces yeasts
(Stellenbosch : Stellenbosch University, 2015-04) Mehlomakulu, Ngwekazi Nwabisa; Divol, Benoit; Setati, Mathabatha Evodia ; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a previous study were identified to strain level and found to belong to the yeast species Candida pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in red wine, and against several strains of the genus Brettanomyces on white and red grape juice medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete killer toxins, which can play a role in yeast microbial interactions under winemaking conditions. The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5 and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during winemaking did not affect the activity and stability of these killer toxins. Although, the killer toxins differed with regards to their biochemical and environmental stability and activity, they were found to have a similar mode of action. The killer toxins induced a fungistatic effect on B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by K. wickerhamii. According to the author’s knowledge this is the first report on the identification of novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed provides great research scope in this field. The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase) and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were identified as the potential toxins, but their killer activity could not be confirmed. These findings suggest that hydrolytic enzymes possess killer activity, as previously reported in literature. However, further investigation is needed to identify the killer toxins characterized in this study.
The impact of UV-light on grapevine berry and wine metabolites.
(Stellenbosch : Stellenbosch University, 2018-12) Honeth, Chandré; Vivier, Melane A.; Young, Philip R.; Stellenbosch University. Faculty of AgriSciences. Dept. Viticulture & Oenology & Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Within their natural environments, plants are constantly challenged by a multitude of stress factors and have therefore evolved different adaptive strategies to mitigate potential damage as well as to optimise resource utilisation. Sunlight, being one of these abiotic factors, is fundamental to plant functioning, however also represents a potential source of stress and damage. Understanding light stress and consequent plant responses have therefore received considerable attention. The impacts of light on plant development have been studied comprehensively in model systems as well as crop plants. As one of the most commercially important fruit crops, grapevine has received considerable attention and significant headway has been made in recent years to profile the potential responses of grapevine tissues to light quantity and quality, specifically UVB. Despite this progress, scope for further exploration into the impacts of varying light quality exposure on berry growth and composition, as well as the extended effects into the wine matrices still exists. The purpose of this study was therefore to examine the impacts of a modulated exposure level (quantity of light) in combination with modulated quality of light (UVB presence or attenuation) on Sauvignon Blanc grape berry growth and metabolite composition during the development and ripening processes, as well throughout the wine-processing steps, ending with a sensorial description of the wines. The distinctive varietal style of Sauvignon Blanc has been well characterised in relation to light exposure, making this cultivar an ideal study system for evaluating the impacts of UVB radiation. The trial was designed using a field-omics approach where an experimental system in a cool-climate Sauvignon Blanc vineyard was previously validated to study berry metabolism under high and low light exposure in the bunch zones. This provided an advantageous base from which to evaluate the grape berry responses to UVB radiation under these two light regimes by strategically installing UVB-attenuating acrylic sheets over the bunch zone, thereby creating the following four distinctive bunch microclimates, namely high light and low light microclimates, which served as the controls for the high and low light -UVB microclimates, respectively. Meso- and microclimatic monitoring confirmed that the intended light conditions were indeed achieved in the various microclimates. When evaluating the high light and low light environments separately, the data confirmed the successful attenuation of UVB in each condition while light exposure remained unaffected by the UVB attenuating sheets. The metabolic responses of the berries under the different microclimates were evaluated by profiling and quantifying primary and secondary metabolites in the whole berries during the developmental and ripening period by sampling at the green, véraison and ripe berry stages over three consecutive seasons. Major sugars and organic acids, photosynthetic pigments, volatile organic compounds, amino acids and polyphenolics were profiled and quantified in the samples and subjected to statistical and multivariate data analyses to reveal developmentally responsive metabolites, and/or metabolites that responded to the variable light quantity and quality exposure level. It was clear that in addition to developmental patterns, variations in exposure and UVB levels lead to particular changes in berry metabolite compositions. The results extended the current understanding of UVB responses in grapevine berries by showing that during the green developmental stage, certain carotenoids implicated in photoprotection responded to the variation in light exposure and that the UVB signal specifically was implicated in the photoinhibition response linked to the violaxanthin cycle. Interestingly, under lower light conditions, a similar UVB dependency was seen for the accumulation of lutein epoxide, a xanthophyll linked to acclimation in shade conditions. The primary metabolites as well as the chlorophylls and major carotenoids were mostly unaffected by UVB radiation, indicating that the berries successfully acclimated to their different microclimates. The metabolic profiles of the photoprotective compounds however suggested that the berries in the UVB attenuation microclimates were possibly more prone to stress, particularly in the low light UVB attenuated environment. The ripe berries also responded to UVB attenuation, but in a different way to the green berries. These responses were furthermore influenced by the level of light exposure. In the ripe berries, the formation of compounds known to have antioxidant and/or “sunscreening” properties were negatively impacted when UVB was attenuated. This was most notable in the high light environment where ambient UVB levels lead to an increase in polyphenolics as well as in certain berry volatile compounds including monoterpenes and norisoprenoids. Similarly, the amino acid composition of the ripe berries was differentially modulated by UVB, specifically regarding the branched chain amino acids and GABA, which may be implicated in the mitigation of stress through their roles as metabolites or signalling compounds. Overall, the results indicated a switch in berry employed acclimation strategies to UVB between the early and late stages of development. The primary objective of the green berries appeared to be the maintenance of photosynthetic activity, whereas the ripe berries shifted their metabolism to accumulate compounds involved in blocking UVB and maintaining the antioxidant status of the tissues as protective measures. The skins and pulp tissues of the ripe berries were also subjected to cell wall profiling techniques, but no indication of altered cell wall monomer or polymer profiles were detected for the different microclimates. The ripe grapes from the four microclimates were used to conduct a grape to juice to wine metabolite profiling analyses, using a typical Sauvignon Blanc vinification work-flow and including a sensory description of the resultant wines. Juice samples were generated at three pre-fermentation processing steps and evaluated for amino acids, polyphenolics, volatile compounds and glutathione. The results firstly confirmed that the four microclimates yielded four unique juice matrices. Secondly, by tracking the metabolites through the three juice possessing steps, evidence of the inherent dynamic nature of the juice matrices was revealed, implying the presence of chemical or biological processes which influence susceptible compounds during processing. Additionally, the variations in both light quantity and quality altered the juice environment by possibly changing the juice oxidation status and the juice lipidome, which also impacted the outcome of certain compounds in the wine. The chemical analysis of the wine focused on the fermentation derived compounds and the results confirmed the significant influence of the microclimate on the chemical compositions of the wines. The most notable impacts were noted in the young wines where a higher content of esters was seen with ambient UVB exposure in both the high light and low light microclimates. These results could potentially be related to amino acid composition of the juices, however significant changes occurred in the finished wines during aging. Sensorial analysis of the final bottled wines following aging revealed perceptible differences associated with the four different microclimates. The results reiterated the characteristic aromatic changes which occur in Sauvignon Blanc wines in relation to the variability of light quantity, but also highlighted the significant impact of specifically UVB on wine sensorial characteristics. In the high light microclimates, ambient UVB exposure was strongly associated with tropical aromatic wines, while the attenuation of UVB generated wine with certain similarities to those of the low light microclimate. This indicated that the UVB component of light was necessary for the formation of compounds responsible for the tropical aromas. Furthermore, the low light microclimate wines were generally described as more green in character, however the attenuation of UVB significantly intensified these aromas. Overall, the results show the significant influence of berry microclimate on grape berry composition, leading to altered juice and wine matrices and ultimately perceivable differences in the wines. The findings of this study therefore provided new insights into the underlying metabolic mechanisms employed by grape berries to acclimate to UVB radiation, revealing the employment of phenotypic plasticity by Sauvignon Blanc. The results furthermore highlighted the influence of UVB on juice and wine compositional properties and also provided novel insights into the grape-juice-wine transitions of certain metabolites in Sauvignon Blanc.
The interaction between Vitis vinifera and fungal pathogens : a molecular approach using characterized grapevine mutants
(Stellenbosch : Stellenbosch University, 2017-03) Moyo, Mukani; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The commercially cultivated grapevine species, Vitis vinifera, is highly susceptible to a wide range of pathogens and pests which include the fungus, Botrytis cinerea. During infection of a wide range of hosts, B. cinerea utilises a combination of cell wall degrading enzymes, phytotoxins and metabolites (amongst others) to facilitate entry into host cells, killing them in the process. Being a necrotroph, B. cinerea feeds off the dead cells and continues to proliferate. One of the lines of defence utilised by plants is through the action of cell wall associated polygalacturonase inhibiting proteins (PGIPs) whose roles include inhibiting the activity of B. cinerea endopolygalacturonases (BcPGs), prolonging the existence of longer chain cell wall fragments involved in signalling and priming the plant prior to infection. The defence roles of grapevine pgip encoding genes (Vvipgip1 from V. vinifera and non-vinifera pgips from wild vines) were previously elucidated in tobacco overexpression studies where they increased resistance to a hyper-virulent B. cinerea strain isolated from grapes. However, overexpressing two of the non-vinifera pgips in V. vinifera conferred the transgenic population with hyper-susceptibility to the same B. cinerea grape strain. This study aimed to comprehensively investigate the basis of the hyper-susceptible phenotype displayed by asking and answering important questions regarding the ability of the non-vinifera PGIPs to interact with and inhibit Botrytis ePGs on the one hand and on the other hand also investigate potentially other (non-ePG inhibition related) functions of the grapevine PGIPs. In silico structural docking simulations of grapevine PGIPs (VviPGIP1 and the two non-vinifera PGIPs) against BcPGs from the grape strain and two other B. cinerea strains (included for comparison) were conducted to gain an understanding of the inhibition interactions from a structural perspective. The predicted PGIP-BcPG interactions were highly B. cinerea strain specific with subtle PGIP genotype specificity. This prompted infection of the transgenic grapevine population with a different B. cinerea strain (B05.10) and the results complemented the in silico docking simulations. The transgenic grapevines did not display hyper-susceptibility to B05.10, indicating that it was a strain specific response. Transgenic tobacco with the same genes overexpressed, on the other hand, displayed increased resistance irrespective of B. cinerea strain used. The phenotype displayed by transgenic grapevine to B. cinerea grape strain infection was thus considered both host and strain specific. Moreover, when B. cinerea mutants in ePGs and galacturonic acid metabolism were used in infection analyses on these grapevine and tobacco populations, both host specific virulence factors and potential recognition/decoy factors could be identified. These results all confirm the importance of the specific host and pathogen and the resulting phenotype and makes it clear that interactome studies would be the most insightful in studying infection/defence. Interestingly, the transgenic grapevine population displayed partial resistance to a biotrophic pathogen, specifically in blocking initial penetration of the pathogen, indicating that the PGIP overexpression could have modulated pre-formed defences in a possible priming mechanism. Further analysis of the transgenic grapevine population confirmed that both the native and transgenic pgips were expressed during infection and active proteins, which effectively inhibited BcPGs, was produced. However, prior to infection, transgenic grapevine leaves displayed a reduction in abundance of cell wall components associated with cell wall strengthening, indicating potential weakened cell walls. Additionally, they emitted significantly lower levels of defence-related sesquiterpenes compared to the controls during B. cinerea grape strain infection. These findings were suggestive of changes in metabolic processes, brought about by overexpressing non-vinifera pgips in V. vinifera background, which favoured the pathogen over the host during infection. Thus to build on this, a whole transcriptomic study to investigate the strain specific infection strategy together with the host specific defence strategy as a dynamic interaction was conducted during the early stages of infection. B. cinerea grape strain expressed significantly higher levels of genes involved in phytotoxin synthesis on transgenic plants compared to the controls, at the local infection site. On the other hand, the transgenic plants expressed significantly lower levels of defence-related genes, also at the local infection site. Taken together, the findings of this study challenge our current understanding of the roles of PGIPs in plant defence during B. cinerea infection. It points towards the possibility that grapevine PGIPs in their native backgrounds are not primarily linked to the classical PGIP-PG fungal inhibition interactions. It also provides insight that the hyper-virulent grape strain possibly optimised mechanisms to use the plant’s defence mechanism against itself and even modulate the host-responses in its favour. The host- and pathogen specific reactions observed in this study strongly highlights the impact that the choice of host-pathogen pairing has on defining defence phenotypes. Future studies should consider strain and host specific responses and interactome approaches would be valuable to that effect. This study successfully characterised the hyper-susceptible phenotype as set out initially, but also provided several new insights as well as new testable hypothesis that can lead to further studies.
Investigating grape berry cell wall deconstruction by hydrolytic enzymes
(Stellenbosch : Stellenbosch University, 2015-04) Zietsman, (Anscha) Johanna Jacoba; Vivier, Melane A.; Moore, John P.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue.
Investigating the impact of MpAPr1, an aspartic protease from the yeast Metschnikowia pulcherrima, on wine properties
(Stellenbosch : Stellenbosch University, 2017-03) Theron, Louwrens Wiid; Divol, Benoit; Bely, Marina; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Protein removal is a key step during the production of white wine in order to avoid the possible appearance of a harmless but unsightly haze. Alternatives to the use of bentonite are actively sought because of technological, organoleptic and sustainable issues associated with its use. Acid proteases that are able to break down proteins under winemaking conditions could be one such alternative. Recent literature reports the successful outcome of the addition of fungal aspartic proteases from Aspergillus and Botrytis. In this study, MpAPr1, an extracellular aspartic protease previously isolated and partially characterised from the yeast Metschnikowia pulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymatic properties of MpAPr1 were initially (Km, Vmax, K’i, optimal pH and temperature for protease activity, impact of minerals, sugars and ethanol on protease activity) characterised in a crude extract. After several attempts using different techniques, MpAPr1 was successfully purified via cation exchange chromatography. Its activity against haze-forming grape proteins was initially tested in a model solution under optimal environmental conditions (for MpAPr1 activity) and under those occurring during winemaking (pH 3.5 and 25°C). Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. These experiments showed that MpAPr1 was able to degrade certain haze-forming proteins, especially chitinases, under optimal conditions and to a lesser extent under winemaking conditions. Prior denaturation of the target proteins by heat treatment was also not required. Moreover, MpAPr1 was able to degrade yeast proteins in a model solution under both conditions. Finally, the presence of MpAPr1, supplemented to grape must, resulted in the partial degradation of grape proteins throughout fermentation and ultimately in a slight difference in the wine’s volatile compound composition. Winemaking conditions limited its impact and it is thus proposed that future work focus on enhancing MpAPr1 activity to make it a viable alternative to bentonite. The study nevertheless provides further evidence that aspartic proteases could represent a potential alternative to bentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrima could have a beneficial impact on wine properties.
Maltotriose transport in yeast
(Stellenbosch : Stellenbosch University, 2007-12) Smit, Annel; Cordero Otero, Ricardo R.; Pretorius, Isak S.; Du Toit, Maret; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries.
Metabolomic profiling of non-Saccharomyces yeasts in wine
(Stellenbosch : Stellenbosch University, 2016-03) Whitener, Margaret Elizabeth Beckner; Du Toit, Maret; Divol, Benoit; Vrhovsek, Urska; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Recent trends in wine making have led to the commercial production and use of non- Saccharomyces yeasts in wine making. Very little is understood however about how the use of these yeasts affects the final product. The purpose of this study was to evaluate the chemical and sensory characteristics of wine fermented with non-Saccharomyces yeasts using a sequential inoculation strategy. Targeted and untargeted analysis techniques were developed to help identify and quantify the volatile fraction of the wines produced. By combining this and sensory data we were able to build the most comprehensive picture to date of the volatile wine metabolome as it is influenced by various yeast species. The first step was a literature review dedicated to summarizing the current knowledge surrounding the metabolomics of the yeasts used in the subsequent chapters. Specifically, we sought to understand what is currently known about the use of non-Saccharomyces yeasts in wine. Also investigated were the technologies currently being used in the fields of food, wine, and yeast metabolomics. The goal was to provide the background necessary to understand the research in the subsequent chapters, as well as aid in the development and planning of the experiments discussed here within. Two stages of research were conducted. Not only did we want to understand the effects of non-Saccharomyces yeasts on wine aroma but we were interested in whether or not these effects were the same in both red and white wines. As such the first research stage, was a preliminary investigation of the yeast response to two different grape musts. Five different species of non- Saccharomyces yeasts, were chosen and grown in both Shiraz and Sauvignon blanc must and samples were collected for analysis just prior to the point at which Saccharomyces cerevisiae would usually be added to complete the fermentation. The fermentation rates were monitored and the chemical profile of the musts was evaluated. A solid-phase microextraction-Gas Chromatography-Mass spectrometry method that targeted 90 different compounds known to be found in wine was used to evaluate the headspace of the fermented musts. The results obtained helped shape the experimental design for the next phase of the project. The scale was increased to full wine production to evaluate how the yeasts could influence a completed wine product. Again, Sauvignon blanc and Shiraz were chosen and an untargeted chemical analysis method was developed to ensure that the widest possible range of analytes could be evaluated. The finished Sauvignon blanc wine was also subjected to sensory analysis which provided even greater insight into how these inoculation strategies can change the sensory profile of the wine. This research was undertaken in an attempt to answer the questions of ‘What will the wine smell and taste like if I use non-Saccharomyces yeasts during fermentation?’ and ‘Could it be superior to standard wines only inoculated with S. cerevisiae?’ The experiments conducted provided a great deal of insight that can help to begin answering these questions but there is much that remains unknown. In general, we were able to build a detailed volatiles chemical profile for each of the yeast treatments used in both Shiraz and Sauvignon blanc. While some treatments proved to be somewhat detrimental to the aroma and flavor of the wine, others showed promise in possibly enhancing its complexity. We were also able to demonstrate that the yeasts behave very differently in the two different musts. As comprehensive as these studies were, future work should be undertaken to improve the understanding of why and how these yeasts can make an impact on wine production. For example, our work did not include any genetic expression analysis of the yeasts used. Correlating genetic expression to quantitative chemical analysis would provide a much more complete picture of the wine yeast metabolome.
Metagenomic screening of cell wall hydrolases, their anti-fungal activities and potential role in wine fermentation
(Stellenbosch : Stellenbosch University, 2015-04) Ghosh, Soumya; Setati, Mathabatha Evodia; Divol, Benoit; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The grape and wine ecosystem contains fungi, bacteria and yeasts whose interactions contribute to the final wine product. While the non-Saccharomyces yeasts are dominant in the early stage of alcoholic fermentation, the later stage is always dominated by Saccharomyces cerevisiae. Although their presence in wine fermentation is often short-lived, the non-Saccharomyces yeasts are known to produce an array of extracellular hydrolytic enzymes which facilitate the extraction and release of aroma compounds, but might also play a role in microbial interactions. The present study aimed to investigate the microbial diversity of grape juice and to evaluate the potential of non-Saccharomyces yeasts to produce hydrolytic enzymes and display anti-fungal properties. To capture the microbial diversity, culture-dependent (plating) and –independent (Automated Ribosomal Intergenic Spacer Analysis (ARISA)) techniques were used in parallel. The fungal and bacterial ARISA displayed a wider range of operational taxonomic units (OTUs) in comparison to cultivation-based technique, demonstrating that ARISA is a powerful culture-independent technique applicable to ecological studies in wine. Some of the uncommon yeast isolates derived from our cultivation-based study were subjected to an enzymatic screening process. Hydrolases, such as chitinases, β-1,4-cellulases, β-1,3-1,6-glucanases, β-glucosidases, pectinases and acid proteases were specifically sought. Most of the yeast isolates exhibited chitinase, β-1,4-cellulase as well as β-1,3-1,6-glucanase activities. Only Metschnikowia chrysoperlae exhibited β-glucosidase activity. We also retrieved the partial chitinase gene sequences from M. chrysoperlae, Pichia burtonii, Hyphopichia pseudoburtonii that exhibited chitinase activity. Among the isolates, Pseudozyma fusiformata exhibited a strong antagonistic activity against the wine spoilage yeasts B. bruxellensis AWRI 1499 and B. anomalus IWBT Y105. Furthermore, we showed that the killer phenotype of P. fusiformata cannot be attributed to a viral encoded dsRNA. Finally, two metagenomic approaches were employed in an attempt to explore the indigenous microbiome in a more holistic manner, where we adopted whole metagenome Roche GS-FLX 454-pyrosequencing and construction of a fosmid library. The whole metagenome sequencing revealed a wide range of hydrolytic enzymes that showed homology to enzymes from different fungal and non-Saccharomyces yeast species. Moreover, the metagenomic library screening resulted in the retrieval of 22 chitinase and 11 β-glucosidase positive fosmid clones originating from yeasts. Two clones of interest, BgluFos-G10 and ChiFos-C21, were subjected to next generation sequencing. BgluFos-G10 revealed 2 ORFs exhibiting homology to glycosyl hydrolase family 16 proteins whereas no ORFs encoding chitinase enzymes could be identified in the ChiFos-C21 clone. However, all the potential ORFs identified exhibited homology to a gene cluster from Clavispora lusitaniae ATCC 42720, suggesting that the cloned DNA fragments belonged to a yeast species closely related to C. lusitaniae or members of the family Metschnikowiaceae. Overall, our study identified a variety of novel hydrolytic enzymes. However, retrieving the full gene sequences of these identified enzymes would be the immediate follow-up of our study. Moreover, the hydrolytic and antifungal activities exhibited by the yeast isolate could be of major interest in evaluating their potential as biocontrol agents against grapevine fungal pathogens and subsequently the wine spoilage yeasts. It would be interesting to evaluate as well the potential impact of these enzymes under wine making condition and could be our next step of investigation.
Molecular analyses of candidate carotenoid biosynthetic genes in Vitis vinifera L.
(Stellenbosch : Stellenbosch University, 2004-03) Young, Philip Richard; Vivier, Melane A.; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole.
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.
A pathogen-derived resistance strategy for the broad-spectrum control of grapevine leafroll-associated virus infection
(Stellenbosch : Stellenbosch University, 2003-12) Freeborough, Michael-John, 1971-; Pretorius, I. S.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco.

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