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.
Cell differentiation in response to nutrient availability : the repressor of meiosis, RME1, positively regulates invasive growth in Saccharomyces cerevisiae
(Stellenbosch : Stellenbosch University, 2003-03) Hansson, Guy Robert, 1974-; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription.
Coevolution of Saccharomyces cerevisiae and Lactobacillus plantarum : engineering interspecies cooperation
(Stellenbosch : Stellenbosch University, 2018-03) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Microbial interactions are ubiquitous in nature and play a vital role in economically important industrial processes like winemaking. Saccharomyces cerevisiae and Lactobacillus plantarum are important species responsible for the completion of alcoholic and malolactic fermentation (AF and MLF) respectively. Understanding how these species interact with each other and their environment is important to better manage successful completion of AF and MLF. However, the complexity of the wine matrix makes it nearly impossible to study these interactions in a natural environment and synthetic ecological systems can therefore be used to overcome these difficulties. This study was designed to establish a co-dependent, mutualistic relationship between S. cerevisiae and Lb. plantarum in order to gain insights into the cooperation between species, how pH, temperature, and inoculation dosages influences the interaction, and how the interaction evolves over time. The interaction, centered on the reciprocal exchange of amino acids, was established between the lysine auxotrophic strain S. cerevisiae THI4 and the isoleucine, alanine, valine, and methionine auxotrophic strain Lb. plantarum B038. Different combinations of amino acids were omitted from the chemically defined synthetic grape juice-like media in order to find an amino acid treatment which promoted the best growth for both microorganisms. B038 showed excellent growth when cocultured with THI4 for all the amino acid treatments, but THI4 struggled to grow under these conditions. The two treatments selected for further experiments were the Lys-Ile (lysine and isoleucine omitted) and Lys-Val (lysine and valine omitted) treatments since THI4 showed the best growth under these conditions. Lower temperature and pH conditions had a negative effect on the growth and malic acid consumption of B038, but when co-cultured with THI4 the yeast appeared to stimulate the growth of the bacteria under both selective and control conditions. THI4 continued to show poor growth performance and sugar consumption under these conditions. However, when THI4 and B038 were inoculated at cell densities with similar biomass, the growth of THI4 improved significantly. It was expected that THI4 and B038 would show poor growth when grown in the absence of their respective auxotrophic amino acids and support of their respective partner. This proved true for all the amino acid treatments except when B038 was grown in the absence of lysine and valine. B038’s ability to grow under these conditions was hypothesized to be linked to the uptake of glutamine and the production of γ-Aminobutyric acid (GABA), but further research is still required to investigate this. Over continuous rounds of fermentation, THI4 adapted to the imposed selective conditions by increasing its consumption of glucose while cell density remained the same. Whether this is linked to increased ethanol production still needs to be determined. No significant changes were observed in B038 after coevolving the strains. This study provides relevant insights into the industrially important interaction between S. cerevisiae and Lb. plantarum and also provides a basis for future work to create optimised yeast-bacteria pairings for both industrial applications in winemaking and to investigate the genetic changes involved in the establishment of cooperative interactions between species.
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.
Engineered yeast and microalgae mutualisms: Synthetic ecology applied to species isolated from winery wastewater
(Stellenbosch : Stellenbosch University, 2018-03) Simpson, Zoe Faith; Bauer, Florian; Naidoo, Rene K.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Large volumes of winery wastewater, classified as biodegradable industrial effluent, are generated annually. The development of a cost-effective treatment system is difficult due to the variable and batch nature of winery wastewater. Research has focused on the development of dynamic biological treatment systems using microorganisms including yeast and microalgae, as it has become apparent that these microbes have bioremediation capabilities in various wastewaters. However, no biological winery wastewater treatment system, employing multi-species ecosystems with known species of yeast and microalgae, currently exists. In this study, yeast and microalgae were previously isolated from natural winery wastewater. The first aim of this study was to characterise the bioremediation potential of the yeast, Saccharomyces cerevisiae, and microalga, Parachlorella beijerinckii, in synthetic and raw winery wastewater. P. beijerinckii was physiologically characterised and was able to tolerate salinity and ethanol levels commonly associated with winery wastewater, making it a suitable candidate for further bioremediation studies. Both S. cerevisiae and P. beijerinckii were able to decrease the chemical oxygen demand of winery wastewater and P. beijerinckii monoculture was able to increase the pH of the acidic wastewater. S. cerevisiae out-competed P. beijerinckii in co-culture growth experiments. Interestingly, yeast growth was improved in the presence of the microalgae in this system, suggesting a potential for symbiotic association. The increased yeast growth however had no impact on the bioremediation potential of the co-culture system. To overcome this drawback, a synthetic ecology approach was used to engineer stable symbiotic associations between these evolutionarily unrelated strains of yeast and microalgae. Engineered mutualisms between S. cerevisiae and P. beijerinckii were established under strongly selective conditions based on the nutrient exchange of carbon and nitrogen. These mutualistic associations were relatively easy to establish as the complementary metabolic abilities of each organism were key elements in the mutualism design. The impact of temperature and pH were assessed in these obligatory mutualistic conditions to determine whether the co-culture functions optimally in specific environmental conditions and whether such conditions are similar or different from the optimal conditions required for single species growth. Experiments were first conducted in small scale and continued in larger scale bioreactor studies. The bioreactor conditions were evaluated to generate a more constant continuous culture system. Such continuous culture system would provide an ideal tool to conduct studies on the evolutionary development of mutualistic associations, and may be the first step in developing a multi-species approach to winery wastewater treatment with enhanced bioremediation capabilities. We propose that in the long run such co-culture systems might serve to overcome the limitations associated with single culture system and might improve biotechnological processes.
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.
Evaluating the impact of yeast co-inoculation on individual yeast metabolism and wine composition
(Stellenbosch : Stellenbosch University, 2014-12) Mains, Arlene Olive; Bauer, Florian; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with Saccharomyces cerevisiae in mixed starter cultures has become an accepted oenological tool to enhance the organoleptic properties of wine. Recent studies have indeed demonstrated the positive contribution that non- Saccharomyces yeasts may have on the bouquet of wine. These mixed starter cultures are characterized by high inoculation levels of individual strains into the must, and each strain in turn is characterized by its own specific metabolic activity. These factors lead to a multitude of interactions occurring between the individual populations within the must. The fundamental mechanisms which drive these interactions are still largely unknown, but several studies have been conducted in order to investigate the metabolic outcome of these interactions. In this study, we endeavour to further characterize the interactions which occur between four individual non-Saccharomyces yeast strains in mixed culture fermentation with S. cerevisiae. Metschnikowia pulcherrima IWBT Y1337, Lachancea thermotolerans IWBT Y1240, Issatchenkia orientalis Y1161 and Torulaspora delbrueckii CRBO LO544 were used in mixed culture fermentations with a commercial strain of S. cerevisiae at an inoculation ratio of 10:1 (non-Saccharomyces: S. cerevisiae). The biomass evolution and fermentation kinetics of both participating species were affected by the high cell density of the other, with neither population reaching the maximal density attained by the pure culture fermentation. The final wine composition of each individual mixed fermentation showed clear differences, from the pure cultured S. cerevisiae and from each other, based on the concentrations of the major volatile compounds found in the wine. Upon further characterization of these specific mixed culture fermentations, it was found that each individual combination of non-Saccharomyces and S. cerevisiae produced similar increases and decreases of certain major volatile compounds as demonstrated by previous authors, using the same combination of non-Saccharomyces species together with S. cerevisiae. From a winemaking perspective, the use of these non- Saccharomyces yeast strains in combination with S. cerevisiae could be a useful strategy to diversify the chemical composition of wine, by increasing the concentration of certain desirable volatile compounds and by modulating the concentration of undesirable metabolites. Furthermore, this research serves as a foundation for further elucidation of the interactions which drive these metabolic outcomes in response to the high cell density of two yeast populations in mixed culture fermentations.
Evaluating the vitamin requirements of wine-related yeasts and the resultant impact on population dynamics and fermentation kinetics
(Stellenbosch : Stellenbosch University, 2019-04) Julies, Jerobiam Marvin; Bauer, Florian; Divol, Benoit; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Within the vineyard environment, grape berries serve as a habitat to various microorganisms including bacteria, filamentous fungi and yeasts, of which some play distinct roles in winemaking. Studies on yeast species other than Saccharomyces cerevisiae, commonly referred to as non-Saccharomyces (NS) yeasts in oenology, have evaluated the ability of these yeast to modulate the sensory profile of wine. In the early stages of spontaneous fermentation when the ethanol concentrations are low, the NS yeast population increases, but is progressively replaced by S. cerevisiae, which is better adapted to the environmental conditions associated with fermenting grape juice. The overall sensory profile of wine is in part a result of the metabolite production of yeasts, and the extent of the contribution of each species will depend on the total metabolic activity of each species. Metabolic activity is directly related to the availability of nutrients such as carbon, nitrogen, vitamins and trace elements. These nutrients are indeed converted to biomass and other metabolites, many of which are aroma and flavour active by-products. Only limited information regarding the nutrient requirements of wine-related yeasts other than S. cerevisiae has been published. Several studies have explored the carbon and nitrogen requirements of some NS species, but the vitamin requirements of many biotechnologically relevant species remains to be determined. Vitamins are organic compounds, mostly of a complex chemical nature, and serve as cofactors in metabolic reactions. Vitamins occur in small quantities in grapes and grape juice, but some data suggest that they may in some cases be limiting for yeast growth in this environment, affecting metabolism and ultimately impact the final wine. This knowledge gap motivates the current study, which focuses on the growth and fermentation kinetics of different NS yeasts when presented with varying concentrations of the relevant vitamins: biotin, pantothenate, inositol, thiamine and pyridoxine. In a first section, a high-throughput microtiter plate assay was optimised to allow for the rapid screening of the vitamin requirements of NS yeasts. The results of this assay showed differences in the vitamin requirements amongst the different yeasts. The statistically most significant vitamin-dependent yeast phenotypes from the screen were selected for further investigation. These included the dependence of Viniflora® P. kluyveri Frootzen ™ on biotin and thiamine and of Viniflora ® L. thermotolerans Concerto ™ on inositol. The data obtained from this study provide a better understanding of the vitamin requirements of NS yeasts and how these requirements can potentially enhance the growth performance of NS yeasts. The data suggest that targeted nutrient additions may lead to a better modulation of the overall sensory profile of wine.
Evolution of mutualistic behaviour between chlorella sorokiniana and saccharomyces cerevisiae within a synthetic environment.
(Stellenbosch : Stellenbosch University, 2020-12) Oosthuizen, Jennifer Rae; Bauer, Florian; Naidoo-Blassoples, Rene Kathleen; Rossouw, Debra; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Microbial symbioses are abundant in the natural environment. Mutualisms are a subset of these symbioses that still lack fundamental understanding regarding the manner in which these complex interactions form and alter microbial species over time. Phototrophic-heterotrophic microbial systems are becoming more commonplace in research due to the many benefits they can provide when different organisms are combined. Heterotrophic fungal systems are largely utilized in the production of high-value metabolites, while phototrophic microalgal systems are found primarily in the green sector such as carbon dioxide sequestration or biofuel production. Synthetic ecology implemented into thoughtfully designed artificial ecosystems provides an ideal method for both the fundamental study of mutualistic symbioses and the production of improved microbial strains for industry. Both the long- and short-term effects of microbial co-evolution on strain performance are largely unknown. Mutualistic interactions are a way to study these effects as the nature of the interaction, reliance on the survival of a partner species, prevents a single species from outcompeting the other. The clear benefits of mutualistic interactions for industrial applications, such as increased growth of both species and/or the production of novel metabolites, also provide clear incentives to investigate these interactions. This study employed synthetic ecology principles and designed an artificial ecosystem to investigate the effects of co-evolution on a mutualistic yeast-microalgal pairing. The yeast, Saccharomyces cerevisiae, and microalga, Chlorella sorokiniana, were co-evolved in an environment that imposed an obligate mutualism between the two microbial partners for approximately 100 generations. The obligate mutualistic interaction was based upon the reciprocal exchange of carbon (CO2 from S. cerevisiae) and nitrogen (ammonia from C. sorokiniana). Strains were isolated from the 50th and 100th generation for further phenotypic, metabolic and transcriptional analysis compared to the parental strains. Phenotypic screening of isolates took place in both mono- and co-culture (multiple pairwise combinations of evolved yeast and microalgae) with various carbon and nitrogen sources to test the limits and effects of co-evolution. This study clearly demonstrated how even short periods of co-evolution can cause changes to the phenotypic growth and metabolite usage of co-evolved isolates. All co-evolved yeast and microalgal strains showed changes to growth rate and a wide variety of growth patterns when compared to the parental strains. Importantly, changes in the expression of key carbon and nitrogen genes were also observed in the evolved isolates of both species. These observed changes assist in highlighting potential underlying mechanisms that occur during co-evolution. These results, when taken together show that even short periods of co-evolution, can produce strains with different characteristics to the parental strains. Harnessing techniques such as co-evolution in combination with synthetic ecology and artificial ecosystems will allow for the creation of functional ecosystems with applications in a wide variety of sustainable industries such as the bioremediation, carbon capture and biofuel industries.
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.
Impact of complex yeast nutrient products on selected non Saccharomyces yeasts
(Stellenbosch : Stellenbosch University, 2020-03-31) Beukes, Louisa; Jolly, Neil; Divol, Benoit; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: In recent years there has been a growing interest in non-Saccharomyces yeasts for winemaking due to their ability to produce more complex wines. These yeasts, considered weak fermenters, are used in combination with Saccharomyces cerevisiae and compete for nutrients such as nitrogen. Therefore, it is important for the winemaker to know what nutrients may be insufficient so that corrective action can be taken. Yeast assimilable nitrogen (YAN), a growth limiting resource naturally occurring in grape must, is important for yeast metabolism as well as for production of desirable aromatic compounds. When YAN is deficient it can lead to slow or stuck fermentations and production of undesirable compounds. Thus, to ensure a complete alcoholic fermentation and desirable aroma profile, nitrogen supplementation is required. Traditionally, ammonium salts are added as a nitrogen supplement, however, recently several complex yeast nutrients have also become commercially available. These yeast nutrients are yet to be investigated for fermentation with non-Saccharomyces yeasts. This study investigated the impact of eight complex commercial yeast nutrients on three commercial non-Saccharomyces yeasts (Torulaspora delbrueckii Biodiva™ TD291, Pichia kluyveri Viniflora® Frootzen™ and Metschnikowia pulcherrima Flavia® MP346). Fermentations were carried out with single yeasts or combined with S. cerevisiae in sequential fermentations in synthetic grape must. The M. pulcherrima sequential fermentation was repeated in Chenin blanc grape must. For the single yeast fermentations, it appeared that the nutrients had a greater effect on the onset of fermentation than on the growth of the yeasts and that one nutrient (nutrient treatment Y2) was preferred by all the yeasts. This is the first time that nitrogen supplementation at the same level but with different content was investigated for non-Saccharomyces wine yeast sequential fermentations. The ability of non-Saccharomyces yeasts to persist in sequential fermentations could be improved with nutrient selection. Further investigations with M. pulcherrima sequential fermentations in Chenin blanc must found clear differences for the two different matrices. Although synthetic must is a defined medium that reduces the risk of unknown variables, it is not a true representation of how these nutrients can influence non-Saccharomyces yeasts in real grape must. Nutrient selection can also increase desirable esters and influence the sensory properties of wine; however, this should be further investigated and confirmed through sensory evaluation. This study improved the current knowledge of non-Saccharomyces yeasts and their utilisation of complex yeast nutrients. It demonstrated that nutrient selection can improve non-Saccharomyces yeast implantation as well as improve production of desirable volatiles.
Insights into South African sparkling wine : a sensory and consumer study
(Stellenbosch : Stellenbosch University, 2020-03) Mokonotela, Tshepo Tshiamo; Nieuwoudt, Helene; Pentz, Chris D.; Jolly, Neil P.; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: To be locally and internationally competitive, the South African wine industry needs to understand consumers’ preferences and purchase motivations. It is especially the sparkling wine category Méthode Cap Classique wines (MCC) that was identified as requiring further insights to develop marketing strategies that appeal to the South African (SA) consumer. This study used an explanatory sequential mixed method design to gain insights into how young (18-35 years old) SA sparkling wine consumer experiences the MCC product offering. A selection of five different styles, namely brut, brut rosé, demi sec, demi sec rosé and low alcohol were chosen. The first objective was to investigate and explore the sparkling wine category producers and growth, as well as the product’s sensory (taste and aroma) characteristics. This was accomplished by data mining of the wine industry information sources Platters South African Wine Guide and the South African Wine Industry Information Systems (SAWIS) database. The second objective investigated how the SA sparkling wine consumer experienced the five styles of MCC intrinsically. This was accomplished by actual tasting of the MCC wines by 278 respondents, who had previously been exposed to MCC or sparkling wine, combined with the completion of a closed-ended tasting questionnaire. The questionnaire included wine evaluation with a Check All That Apply (CATA) list where respondents had to select sensory attributes perceived by them, liking and likelihood to buy, price perception, preference ranking, familiarity with MCC, and product involvement. The third objective investigated young SA sparkling wine consumers’ familiarity with MCC, and their perceptions of the product’s extrinsic features, such as label design, bottle shape and colour, brand name, price, and packaging. This third investigation was done by means of using a semi-structured open-ended questionnaire in one-to-one interviews with 13 consumers who also participated in the tasting.Results of the quantitative phase showed that the respondents indicated an above average liking of the MCC wines tasted. They can distinguish between different styles of MCC in a blind tasting. The respondents liked the sweeter demi sec style the most, and the low alcohol style the least. The respondent’s price perceptions of the wines, based on intrinsic features, were lower than the actual retail prices; however, the price was perceived to be higher than retail prices when based it on the bottle extrinsic features. The respondents also rated themselves as more familiar with sparkling wine than MCC. In the subsequent qualitative approach, the respondent’s familiarity with MCC revealed that they possessed knowledge of the MCC and engaged with the product category. They find different occasions and opportunities to consume MCC that were not limited to formal celebrations, such as weddings or graduations. While occasion of usage is important, bottle appearance and price emerged as the main determinants of whether they would select a bottle of MCC for a specific occasion. Respondents were also aware that their choice of a bottle of MCC for a specific occasion, communicates non-verbal cues about them in a social context to their peers.This study has laid a foundation on how a selection of MCC wines are perceived in South Africa from respondents’ multiple perspectives. Although the study is not generalisable to the whole South Africa, the mixed method research strategy used provided insights into consumers’ perceptions and the use of mixed methods. Of these the preference for sweeter styles and nonpreference for lower-alcohol wines – these aspects can be investigated in follow up studies using the methodology established in this study.
Interaction of multiple yeast species during fermentation
(Stellenbosch : Stellenbosch University, 2015-04) Luyt, Natasha Alethea; Bauer, Florian; Divol, Benoit; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with the yeast S. cerevisiae in multistarter wine fermentations has emerged as a useful tool to modulate wine aroma and/or to decrease the concentration of undesirable compounds. However, upon inoculation, these yeast species do not co-exist passively, but interact in various ways. While competition for nutrients and the excretion of killer toxins in an antagonistic relationship are obvious and well established types of interactions, some studies have suggested the existence of other forms of cellular or molecular interactions. One of these includes physical cell-cell contact and to our knowledge, only one previous study has confirmed its existence in wine yeasts. Yeast interactions are also influenced by other factors, such as ethanol concentration, however some studies have highlighted the role that dissolved oxygen plays on the survival of non-Saccharomyces yeasts and their ability to compete for space with S. cerevisiae and little research has focused on this. This study aimed to investigate the occurrence of a physical cell-cell and/or metabolic interaction between S. cerevisiae and L. thermotolerans in mixed culture fermentations of synthetic grape must. For this purpose, fermentations in a Double Compartment Bioreactor (DCB) which separates yeast population through the use of a membrane were compared to mixed fermentations in the absence of the membrane, using the same reactor. Furthermore, the impact of oxygen supply on yeast behaviour was also assessed. Following mixed culture fermentations in a DCB, it was observed that the presence of S. cerevisiae led to a significant decline in viability in L. thermotolerans. This decline was significantly less prominent in mixed cultures where the cells were in indirect contact. Together, the data provided evidence for both cell-cell and metabolic interactions whereby S. cerevisiae had a strong negative influence on the growth of L. thermotolerans. However, it was also observed that L. thermotolerans had some negative impact on the growth of S. cerevisiae, leading to a reduction in biomass (when in indirect contact) and a reduced maximum CFU/mL compared to pure cultures. The data also suggest that direct physical contact may increase the production of glycerol and propanol, but this needs further investigation. By decreasing the frequency at which oxygen pulses were provided, a reduction in biomass and increase in fermentation duration was observed for all fermentations. However, this effect was somewhat reduced in mixed cultures. Here, no impact on fermentation duration was observed and the decrease in biomass was less compared to pure cultures. The impact of these oxygen pulses was also greater on L. thermotolerans. In the latter yeast’s pure culture a slight increase in glycerol was observed when less oxygen was provided and in general there appeared to be no impact on acetic acid production. Furthermore, there was little or no impact on volatile production, however, more repeats might reveal different results and therefore more research is needed to confirm these results. To our knowledge, this is the first study of its kind to confirm a physical cell-cell interaction between the yeast pair S. cerevisiae and L. thermotolerans.
Investigating osmotic stress in mixed yeast cultures and its effects on wine composition
(Stellenbosch : Stellenbosch University, 2015-04) De Kock, Marli Christel; Divol, Benoit; Bauer, Florian; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Grape must gives rise to various stress conditions for the yeast inoculated for alcoholic fermentation. These include hyperosmotic stress due to the high initial sugar concentration and redox imbalances due to the fast depletion of oxygen. Under these stress conditions, Saccharomyces cerevisiae tends to produce glycerol as an osmoprotectant and to regenerate reducing equivalents. However, the production of glycerol often leads to increased acetic acid production. According to literature, it seems that many non-Saccharomyces yeasts have a different metabolic response to the above-mentioned stress conditions, especially since it has been found that they produce low levels of acetic acid. Only recently non-Saccharomyces yeasts were researched to be used as starter cultures in wine fermentations. It is found that they can confer beneficial characteristics to the resulting wine. However, most of the non-Saccharomyces yeasts lead to stuck fermentations as confirmed by this study. Therefore, if the positive characteristics of these yeasts were to be exploited in wine making they need to be inoculated together with S. cerevisiae. When two yeasts are inoculated together, they affect each other and consequently the wine. In this context, the aim of this study was to investigate the metabolic response to hyperosmotic stress during wine fermentation of the following wine-related non-Saccharomyces yeasts: Lachancea thermotolerans, Torulaspora delbrueckii and Starmerella bacillaris. Fermentations were performed in a synthetic grape must medium with pure cultures of the mentioned strains as well as mixed cultures of each non-Saccharomyces yeast with S. cerevisiae. The fermentation behaviour was monitored and concentrations of various wine-related metabolites were determined. Concerning polyol concentrations, S. cerevisiae produced only glycerol while the non-Saccharomyces yeasts also produced other polyols. The low production of acetic acid in the non-Saccharomyces fermentations was confirmed especially in the case of L. thermotolerans. Moreover, this yeast produced high levels of the higher alcohols butanol and propanol. St. bacillaris produced significant levels of acetoin and isobutyric acid and T. delbrueckii produced an increased concentration of succinic acid. All these metabolites might play a role in maintaining intracellular redox balance. However, a more extensive systematic study is needed to investigate the extent of their involvement. The mixed cultures completed fermentation and had higher final glycerol levels than the control and lower acetic acid concentrations and therefore can contribute positively to the wine aroma. Furthermore, the mixed culture fermentations showed the potential of lowering the ethanol concentrations of wine. Furthermore it has been shown in literature that the yeasts present in the mixed culture can affect each other on gene expression level as well. However, there is little genetic information available on non-Saccharomyces yeasts. In this study, we sequenced the genes involved in glycerol and acetic acid biosynthesis of L. thermotolerans and T. delbrueckii. The gene sequences are fairly homologous with only a few differences. These gene sequences can be used to study gene expression of GPD1 and ALD6 from fermentation samples in order to determine to what extent the yeasts in a mixed culture influence the gene expression of one another.
Lactobacillus plantarum : amino acid utilization
(Stellenbosch : Stellenbosch University, 2018-03) Botma, Izak Johannes; Du Toit, Maret; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Amino acid metabolism serves as a source of sulphur, carbon and nitrogen for bacteria growing in wine or grape juice. The metabolism of amino acids controls the rate of growth and malic acid degradation and also results in the formation of various aromatic compounds which may positively or negatively influence the aroma profile of wine. L. plantarum, a lactic acid bacterium (LAB), may be used as co-inoculant in high pH (≥ 3.5) grape juice for fast malic acid degradation and high aroma production. Since the research on L. plantarum nitrogen metabolism is scarce, the overall goal of this study was to better understand it. The first aim was to determine the amino acid requirements in L. plantarum for growth and malic acid degradation, through single amino acid omissions. This entailed inoculation of nitrogen starved L. plantarum strains into chemically defined media (in this case synthetic grape juice) in which one amino acid is removed at a time. The data suggests that amino acid trophic requirements in L. plantarum are highly strain dependent, although Leu, Ile, Val, Glu and Met were shown under our conditions to be essential amino acids and Gln, Gly, His, Lys and Trp were non-essential amino acids. In a subsequent experiment, 5 single amino acid omissions (Ala, Arg, Gln, Trp and Val) were selected to evaluate their effect on growth and malic acid uptake in synthetic grape juice. During malolactic fermentation (MLF) the removal of Ala and Val had completely repressed MLF induced by L. plantarum while the removal of Trp and Arg had somewhat repressed MLF. Only the removal Gln did not hinder MLF for at least one strain. The second aim was to determine the order of amino acid uptake by L. plantarum in synthetic grape juice using HPLC. Asp, Thr, Ser and Ala tends to be assimilated at a high rate within the first 72 h while the branched chain amino acids, aromatic amino acids (AAA) and Met are assimilated after 72 h. The third aim determined the amino acid uptake in Chardonnay grape juice. The assimilation pattern differed considerably between synthetic grape juice and Chardonnay grape juice. In contrast to synthetic grape juice Arg, Leu, Phe and Ala were preferred amino acid sources. It is thought that the differences could be attributed to mainly two factors: initial nitrogen concentration (40 mg N/L in SGJ vs 240 mg N/L in grape juice) and the pre-culture conditions. This study confirmed that higher nitrogen concentrations resulted in higher growth and quicker malic acid degradation. The high nitrogen requirement of certain amino acids combined with the harsh wine parameters experienced in sequential MLF might explain why L. plantarum struggles with MLF in this scenario. Further research should be directed towards identifying the preferred amino acids in dried and fresh L. plantarum starter cultures to assess if there is a difference. If nitrogen requirements continues to be investigated in L. plantarum successful tailored supplements can be created to aid the growth of L. plantarum in wine or grape juice.
A microbiological solution to visible wine defects : pinking and protein haze formation
(Stellenbosch : Stellenbosch University, 2019-04) Kufa, Amanda; Bauer, Florian; Ndlovu, Thulile; Van Jaarsveld, Francois; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The wine industry is challenged with visible and nonvisible wine defects, which result in profit losses as consumers reject such wines. The clarity and color of the wine are essential to white wine consumers, as it is a measure of quality. Pinking of white wine and the formation of protein haze are some of the most frequent visual defects encountered in the industry. Pinking is a non-scientific term that describes the change in the natural white wine colour to a pink colour. Many speculations have been made concerning the causes of white wine pinking, but there is no conclusive explanation for the phenomenon yet. Protein haze in white wine is caused by the precipitation of pathogenesis-related proteins, namely thaumatin-like proteins, and chitinase. While bentonite is commonly used as a fining agent to avoid protein haze, it has an adverse effect on wine quality. There is, therefore, a need for cost-effective alternatives aimed at preventing wines from both pinking and protein haze formation. Previous studies have reported that some yeast strains have the capability of reducing protein haze formation while no studies to date have reported the impact of yeast strains on wine pinking. In this study, a microbiological based solution was explored and the use of Saccharomyces cerevisiae and other wine-related yeast species as alternatives to chemically based fining agents was investigated. Monocultured and sequentially inoculated fermentations were carried out in both Sauvignon blanc must and synthetic grape must, and yeast cell wall chitin and mannoproteins levels were monitored during fermentation. Interestingly, yeast cell wall chitin and mannoproteins levels decreased by more than half at the end of alcoholic fermentations from the initial day 1 level. A very promising correlation was obtained between chitin in the yeast cell wall and the binding of GFP-tagged chitinase to the cells. Different stains showed different binding affinities, which could be used to predict the haze protection of a particular strain. Some impact of yeast strains on pinking was also observed. In conclusion, the data suggest that yeast strain selection may help reduce, if not in some cases eliminate the need for the use of bentonite as a fining agent for protein haze protection. The data also suggest that pinking can be somewhat reduced in similar ways.
Molecular typing of wine yeasts : evaluation of typing techniques and establishment of a database
(Stellenbosch : Stellenbosch University, 2012-03) Hoff, Justin Wallace; Du Plessis, H. W.; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The yeast species, Saccharomyces cerevisiae and S. bayanus are well known for the key role they play during alcoholic fermentation in both wine and beer industries. These yeasts are available in pure active dried form and can be used to produce different wine styles and to manage quality. There are more than 200 commercial wine yeast strains on the market and include naturally isolated strains and hybrids. With all these commercial yeasts available, strain authenticity is very important to the manufacturer of active dried wine yeasts (ADWY) because it can prevent commercial losses and maintain market credibility. It is as important to the winemaker as it may impact wine quality. Various traditional and molecular techniques have been successfully applied to perform quality control of wine yeast strains. The aims of this study were to evaluate electrophoretic karyotyping (CHEF) and PCRbased methods to distinguish between Saccharomyces wine yeast strains and to establish a database containing molecular profiles of commercial strains. CHEF karyotyping was chosen because it is generally used in the wine industry to distinguish between wine yeast strains, but can be time-consuming. Alternatively, PCR-based methods are considered to be reliable and fast. These PCR methods included the evaluation of interdelta regions, multiplex-PCR of miniand microsatellites, MET2 gene RFLP analysis and the use of several species-specific primers. In this study, 62 commercial wine yeast strains, were randomly selected from various manufacturers of ADWY, and two reference strains, S. bayanus CBS 380 and S. cerevisiae CBS 1171, were evaluated. CHEF karyotyping could successfully differentiate between all 64 yeast strains. The two primer sets used for interdelta amplifications, delta1-2 and delta12-21, yielded 59 and 62 profiles, respectively. Yeast strains considered to be similar or identical according to interdelta amplification results, were resolved with CHEF karyotyping. CHEF karyotyping was proven to be more accurate than interdelta amplifications in distinguishing between commercial wine yeast strains. However, the results of interdelta amplifications were very useful and less time-consuming. The multiplex-PCR of mini- and microsatellite primers only succeeded in identifying a specific band within 55 of the 64 yeast strains including the S. cerevisiae reference strain, a possible indication of species specificity. However, oenological designation using MET2 gene RFLP analysis and species-specific primers indicated that all the commercial strains in this study had a S. cerevisiae ancestry. Restriction analysis of the MET2 gene with EcoRI also successfully identified AWRI Fusion and Zymaflore X5 as hybrid yeast strains. A wine yeast database was created and contains three libraries, i.e. CHEF karyotypes, delta1-2 and delta12-21 electrophoretic profiles. The database was proven to be functional and showed great accuracy in grouping and identifying test strains. The database has many possible applications, but there is still some optimisation and refinement needed.
Mss11p mediated regulation of transcription, pseudohyphal differentiation and flocculation in Saccharomyces cerevisiae
(Stellenbosch : Stellenbosch University, 2004-03) Franken, Jaco (Cornelius Jacobus); Bauer, Florian; Pretorius, I. S.; Patterton, H. G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: In all cellular systems the ability to alter eellular programs in response to extracellular cues is essential for survival. This involves the integration of signals triggered by membrane bound receptors in order to adjust the expression of target genes and enzyme activities and consequently phenotypic outcome. The yeast Saccharomyces cerevisiae has evolved several adaptations, such as, sporulation and pseudohyphal differentiation, in order to survive changes in the surrounding environment. Pseudohyphal differentiation and the related phenotype, invasive growth, are proposed to be adaptations that enable the yeast to forage for scarce nutrients or escape from a detrimental environment. This dimorphic transition is associated with a change from the normal "yeast" form to a pseudohyphal form, which involves changes in budding pattern, cell-cycle progression, cellular elongation, and cell-eell and cell-substrate adherence. The outcome of these changes is elongated eells, which bud in a unipolar fashion and do not separate after budding to form chains of cells referred to as pseudohyphae. These pseudohyphae are able to penetrate the surface of agar containing growth medium, a process referred to as invasive growth. Nutrient-induced adaptations, such as pseudohyphal growth, have been extensively studied in S. cere visiae , and several factors have been implicated in the regulation thereof, many of which are part of specific signalling pathways. The most clearly defined are the filamentous growth specific MAP kinase cascade and the Gpa2p-cAMP-PKA pathway. MUC1/FL011, encoding a member of a family of cell wall associated proteins involved in cellcell/ cell-substrate adhesion, is regulated by these pathways and considered to be critical in the establishment of pseudohyphal differentiation and invasive growth. The promoter region of MUC1/FL011 represents one of the largest yeast promoters identified to date, with cis-acting elements present up to 2.4 kb upstream from the first coding triplet. The upstream regulatory region of MUC1/FL011 is almost identical to that of the STA2 gene, which encodes an extracellular glucoamylase required for the utilisation of extracellular starch. As suggested by the extent of homology between these two promoters, MUC1/FL011 and STA2 are co-regulated to a large degree and both require the same transcription factors. Mss11p plays a central role in the regulation of MUC1/FL011 and STA2 and consequently starch metabolism and pseudohyphaI differentiation. The regulation conferred by MSS11 on the transcriptional levels of MUC1/FL011 and STA2 also appears to be dependent on signals generated specifically in the presence of low nitrogen and glucose. Mss11p does not have significant homology to any other yeast protein, with the exception of limited homology to the transcriptional activator F108p. However, several distinctive domains have been identified in the MSS11 gene product. Firstly, Mss11p contains polyglutamine and poly-asparagine domains. It also contains a putative ATP- or GTP-binding domain (P-Ioop), commonly found in proteins such as kinases, ATPases or GTPases. Two short stretches close to the N-terminal, labelled H1 and H2, share significant homology to the transcriptional activator, F108p. Both the H2 domain and the extreme C-terminal of Mss11p are able to stimulate RNA polymerase II dependent transcription. Furthermore, the H1 domain together with the P-Ioop negatively regulates the activation potential of the H2 domain. This study presents further insight into the functioning of Mss11p and the involvement of the separate activation and regulatory domains in mediating transcriptional activation and pseudohyphal differentiation in response to nutrient limitation. Genetic interactions between Mss11p and other factors involved in the regulation of pseudohyphal growth and starch degradation were revealed, and specific regions of Mss11p were shown to be required by these factors in order to achieve their required function. In addition, results obtained in this study implicates Mss11p in the regulation of Ca2+-dependent flocculation and suggest that the FL01 gene is also regulated by Mss11p in this capacity.
Multispecies interactions in a simplified wine yeast consortium
(Stellenbosch : Stellenbosch University, 2018-03) Nutt, John; Bauer, Florian; Setati, Mathabatha Evodia; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Over the last two decades various studies have evaluated the impact of non-Saccharomyces (NS) yeasts on alcoholic fermentation of wine, the chemical imprint they leave in the wine and how this affects wine quality. These NS yeasts are either present naturally in the grape must or inoculated together with Saccharomyces cerevisiae, but little is known about how these NS interact with each other and with S. cerevisiae and how these interactions might influence the presences of other yeast during fermentation and ultimately affect the contribution of each yeast to the wine. In recent years, several strains of the species, Torulaspora delbrueckii, Pichia kluyveri, Metschnikowia pulcherrima and Lachancea thermotolerans have been commercialized. The availability of such commercial preparations allows winemakers to perform mixed-culture fermentations through different inoculation strategies. Research has evaluated co-inoculations and sequential inoculations between NS and S. cerevisiae, but grape must is a complex ecosystem with a large variety of indigenous yeasts that partake during fermentation. Understanding how various yeasts interact within such a larger matrix is challenging, but will make an essential contribution to sound decision making in wineries. This study was designed to better understand how NS yeasts perform individually and how their behaviour might differ in the presence of one or more other yeasts and what effect this might have on the final wine. Three commercial NS yeasts strains and one S. cerevisiae yeast strain were used to determine how these yeasts interact and how these interactions might alter the chemical composition of wine. Fifteen inoculations scenarios, including mono-culture, co-culture and combinations of three and ultimately a consortium containing all four yeasts were performed. Fermentations were carried out in synthetic grape juice at both 15°C and 25°C. The data showed significant variations in the cell densities of all species through-out fermentation depending on the nature of the co-inoculated species and the environmental conditions. These changes in population dynamics also had a clear impact on the concentration of and types of aromatic compounds produced. Chenin blanc wines made with the consortium of all four yeasts, S. cerevisiae and spontaneous fermentations, showed distinct chemical profiles. However, no correlation was found, regarding population dynamics or aroma profiles of the wines, between the synthetic wine and the Chenin blanc wines both derived from the consortium inoculation. This study provides the foundation for future work on understanding how multiple species (more than two yeasts) interact within fermentations and how this will affect wine quality. It also provides a better understanding of how one yeast can suppress the presence of other yeasts and how different temperatures might affect the presence of each yeasts and how this might influence the interactions between the yeasts.
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.

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