Institute for Wine Biotechnology
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Browsing Institute for Wine Biotechnology by Author "Bauer, Florian"
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- ItemBiosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae(PLoS, 2013) Franken, Jaco; Brandt, Bianca A.; Tai, Siew L.; Bauer, FlorianLevans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.
- ItemCarnitine requires choline to exert physiological effects in saccharomyces cerevisiae(Frontiers Media, 2018-07-02) Du Plessis, Michelle; Franken, Jaco; Bauer, Florian; De Biase, DanielaL-Carnitine is a key metabolite in the energy metabolism of eukaryotic cells, functioning as a shuttling molecule for activated acyl-residues between cellular compartments. In higher eukaryotes this function is essential, and defects in carnitine metabolism has severe effects on fatty acid and carbon metabolism. Carnitine supplementation has been associated with an array of mostly beneficial impacts in higher eukaryotic cells, including stress protection and regulation of redox metabolism in diseased cells. Some of these phenotypes have no obvious link to the carnitine shuttle, and suggest that carnitine has as yet unknown shuttle-independent functions. The existence of shuttle-independent functions has also been suggested in Saccharomyces cerevisiae, including a beneficial effect during hydrogen peroxide stress and a detrimental impact when carnitine is co-supplemented with the reducing agent dithiothreitol (DTT). Here we used these two distinct yeast phenotypes to screen for potential genetic factors that suppress the shuttle independent physiological effects of carnitine. Two deletion strains, Δcho2 and Δopi3, coding for enzymes that catalyze the sequential conversion of phosphatidylethanolamine to phosphatidylcholine were identified for suppressing the phenotypic effects of carnitine. Additional characterisation indicated that the suppression cannot be explained by differences in phospholipid homeostasis. The phenotypes could be reinstated by addition of extracellular choline, but show that the requirement for choline is not based on some overlapping function or the structural similarities of the two molecules. This is the first study to suggest a molecular link between a specific metabolite and carnitine-dependent, but shuttle-independent phenotypes in eukaryotes.
- ItemDetermining the impact of industrial wine yeast strains on organic acid production under white and red wine-like fermentation conditions(South African Society for Enology and Viticulture, 2015) Chidi, B. S.; Rossouw, D.; Buica, A. S.; Bauer, FlorianOrganic acids are a major contributor to wine flavour and aroma. In the past, the scientific focus has mostly been on organic acids derived from grapes or on the transformation of malic acid to lactic acid by lactic acid bacteria, since these acids contribute significantly to the final total acidity of wine. However, the organic acid concentration and composition also change significantly during alcoholic fermentation, yet only limited information regarding the impact of different yeast strains on these changes has been published. Here we report on changes in organic acid (malic, tartaric, citric, succinic, acetic and pyruvic) composition during fermentation by five widely used industrial wine yeast strains in a synthetic grape must (MS300) reflecting two very different, but both wine-like, fermentation conditions. Samples were obtained from three physiological stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). These different stages were selected to provide more information on acid evolution throughout fermentation, as well as on the impact of nutritional and environmental conditions during aerobic and anaerobic fermentation. Among other observations, some strains (such as VIN13 and 285) were shown to be generally higher producers of most acids in white and/ or red wine fermentation settings, while other strains (such as DV10) were generally lower acid producers. The data clearly demonstrate that different strains have different acid consumption and production patterns, and this presents a first step towards enabling winemakers to appropriately select strains for acid management during fermentation.
- ItemThe diversity and dynamics of indigenous yeast communities in grape must from vineyards employing different agronomic practices and their influence on wine fermentation(South African Society for Enology and Viticulture, 2015) Bagheri, Bahareh; Bauer, Florian; Setati, M. E.The current study evaluated the diversity of yeast species in Cabernet Sauvignon grape must derived from three neighbouring vineyards from a similar terroir but on which significantly different management practices are employed. The fermentation kinetics and yeast population dynamics were monitored from the beginning to the end of spontaneous fermentation. The grape musts were characterised by distinct yeast populations comprising oxidative, weakly fermentative and strongly fermentative yeasts. Different combinations of dominant non-Saccharomyces yeasts were observed in each must, with significantly different assortments of dominant species, including Starmerella bacillaris (synonym Candida zemplinina), Lachancea thermotolerans, Hanseniaspora uvarum, Candida parapsilosis and Wickerhamomyces anomalus. None of these yeast consortia appeared to affect the growth of Saccharomyces cerevisiae or inhibit the overall progress of fermentation. However, the percentage of fermentative yeasts was positively correlated with the fermentation rate. Glucose and fructose consumption rates suggested active participation of both glucophilic and fructophilic yeasts from the onset of fermentation. The data highlight two parameters, viz. initial cell concentration and yeast community composition, as important fermentation drivers and open the possibility to predict fermentation behaviour based on the initial composition of the yeast community.
- ItemIdentifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols(Springer-Verslag, 2011-05) Styger, Gustav; Jacobson, Dan; Bauer, FlorianDuring alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae.
- ItemThe impact of single amino acids on growth and volatile aroma production by Saccharomyces cerevisiae strains(Frontiers Media, 2017) Fairbairn, Samantha; McKinnon, Alexander; Musarurwa, Hannibal T.; Ferreira, Antonio C.; Bauer, Florian; Lanciotti, RosalbaNitrogen availability and utilization by Saccharomyces cerevisiae significantly influence fermentation kinetics and the production of volatile compounds important for wine aroma. Amino acids are the most important nitrogen source and have been classified based on how well they support growth. This study evaluated the effect of single amino acids on growth kinetics and major volatile production of two phenotypically different commercial wine yeast strains in synthetic grape must. Four growth parameters, lag phase, maximum growth rate, total biomass formation and time to complete fermentation were evaluated. In contrast with previous findings, in fermentative conditions, phenylalanine and valine supported growth well and asparagine supported it poorly. The four parameters showed good correlations for most amino acid treatments, with some notable exceptions. Single amino acid treatments resulted in the predictable production of aromatic compounds, with a linear correlation between amino acid concentration and the concentration of aromatic compounds that are directly derived from these amino acids. With the increased complexity of nitrogen sources, linear correlations were lost and aroma production became unpredictable. However, even in complex medium minor changes in amino acid concentration continued to directly impact the formation of aromatic compounds, suggesting that the relative concentration of individual amino acids remains a predictor of aromatic outputs, independently of the complexity of metabolic interactions between carbon and nitrogen metabolism and between amino acid degradation and utilization pathways.
- ItemInvestigating the effect of selected non-saccharomyces species on wine ecosystem function and major volatiles(Frontiers Media, 2018-11-13) Bagheri, Bahareh; Zambelli, Paolo; Vigentini, Ileana; Bauer, Florian; Setati, Mathabatha Evodia; Cao, MingfengNatural alcoholic fermentation is initiated by a diverse population of several non-Saccharomyces yeast species. However, most of the species progressively die off, leaving only a few strongly fermentative species, mainly Saccharomyces cerevisiae. The relative performance of each yeast species is dependent on its fermentation capacity, initial cell density, ecological interactions as well as tolerance to environmental factors. However, the fundamental rules underlying the working of the wine ecosystem are not fully understood. Here we use variation in cell density as a tool to evaluate the impact of individual non-Saccharomyces wine yeast species on fermentation kinetics and population dynamics of a multi-species yeast consortium in synthetic grape juice fermentation. Furthermore, the impact of individual species on aromatic properties of wine was investigated, using Gas Chromatography-Flame Ionization Detector. Fermentation kinetics was affected by the inoculation treatment. The results show that some non-Saccharomyces species support or inhibit the growth of other non-Saccharomyces species in the multi-species consortium. Overall, the fermentation inoculated with a high cell density of Starmerella bacillaris displayed the fastest fermentation kinetics while fermentation inoculated with Hanseniaspora vineae showed the slowest kinetics. The production of major volatiles was strongly affected by the treatments, and the aromatic signature could in some cases be linked to specific non-Saccharomyces species. In particular, Wickerhamomyces anomalus at high cell density contributed to elevated levels of 2-Phenylethan-1-ol whereas Starm. bacillaris at high cell density resulted in the high production of 2-methylpropanoic acid and 3-Hydroxybutanone. The data revealed possible direct and indirect influences of individual non-Saccharomyces species within a complex consortium, on wine chemical composition.
- ItemModifying Saccharomyces cerevisiae adhesion properties regulates yeast ecosystem dynamics(American Society for Microbiology, 2018-10-24) Rossouw, D.; Meiring, Skye P.; Bauer, FlorianPhysical contact between yeast species, in addition to better-understood and reported metabolic interactions, has recently been proposed to significantly impact the relative fitness of these species in cocultures. Such data have been generated by using membrane bioreactors, which physically separate two yeast species. However, doubts persist about the degree that the various membrane systems allow for continuous and complete metabolic contact, including the exchange of proteins. Here, we provide independent evidence for the importance of physical contact by using a genetic system to modify the degree of physical contact and, therefore, the degree of asexual intraspecies and interspecies adhesion in yeast. Such adhesion is controlled by a family of structurally related cell wall proteins encoded by the FLO gene family. As previously shown, the expression of specific members of the FLO gene family in Saccharomyces cerevisiae dramatically changes the coadhesion patterns between this yeast and other yeast species. Here, we use this differential aggregation mediated by FLO genes as a model to assess the impact of physical contact between different yeast species on the relative fitness of these species in simplified ecosystems. The identity of the FLO gene has a marked effect on the persistence of specific non-Saccharomyces yeasts over the course of extended growth periods in batch cultures. Remarkably, FLO1 and FLO5 expression often result in opposite outcomes. The data provide clear evidence for the role of physical contact in multispecies yeast ecosystems and suggest that FLO gene expression may be a major factor in such interactions. IMPORTANCE The impact of direct (physical) versus indirect (metabolic) interactions between different yeast species has attracted significant research interest in recent years. This is due to the growing interest in the use of multispecies consortia in bioprocesses of industrial relevance and the relevance of interspecies interactions in establishing stable synthetic ecosystems. Compartment bioreactors have traditionally been used in this regard but suffer from numerous limitations. Here, we provide independent evidence for the importance of physical contact by using a genetic system, based on the FLO gene family, to modify the degree of physical contact and, therefore, the degree of asexual intraspecies and interspecies adhesion in yeast. Our results show that interspecies contact significantly impacts population dynamics and the survival of individual species. Remarkably, different members of the FLO gene family often lead to very different population outcomes, further suggesting that FLO gene expression may be a major factor in such interactions.
- ItemModulation of yeast-derived volatile aromas by oleic acid and sterols(South African Society for Enology and Viticulture, 2019) Fairbairn, S.; Ferreira, A. C. Silva; Bauer, FlorianUnsaturated fatty acids and sterols are essential constituents of the yeast plasma membrane. Recently, their contribution to modulating the production of yeast-derived volatile compounds has received significant attention. The objective of this study was to determine how sterol and lipid supplementation, including ergosterol, plant sterols or oleic acid, differentially influenced yeast growth as well as the production of fermentative aromas when added individually or in combinations. Oleic acid significantly altered the volatile profiles produced and lowered yeast growth. Generally, phytosterol (β-sitosterol) and ergosterol supplementation resulted in similar responses regarding the production of aromas, however, they differed in the magnitude of the response in the case of medium chain fatty acids and acetate esters synthesis. The combinations of sterols with oleic acid resulted in a response more closely associated with the oleic acid control treatment, showing lower levels of acetate ester production.
- ItemNutrient exchange of carbon and nitrogen promotes the formation of stable mutualisms between chlorella sorokiniana and saccharomyces cerevisiae under engineered synthetic growth conditions(Frontiers Media, 2019-04) Naidoo, Rene K.; Simpson, Zoe F.; Oosthuizen, Jennifer R.; Bauer, FlorianMicrobial biotechnological processes can be based on single species pure cultures or on multi-species assemblages. While these assemblages can be advantageous by offering more functionalities and more resilience to changing environmental conditions, they can be unpredictable and difficult to control under synthetically engineered growth conditions. To overcome the unpredictable nature of these microbial assemblages, the generation of stable mutualistic systems through synthetic ecology approaches may provide novel solutions for understanding microbial interactions in these environments. Here we establish a stable association between two evolutionarily unrelated, but biotechnologically complementary species isolated from winery wastewater; a strain of the yeast Saccharomyces cerevisiae and microalga, Chlorella sorokiniana. Yeast and microalgae were able to form obligate (interdependent) and non-obligate (facultative) mutualisms under engineered batch co-culture growth conditions. Obligate mutualism was maintained through the reciprocal exchange of carbon and nitrogen where the yeast ferments mannose to produce carbon dioxide for use by the microalga; and the microalga provides the yeast with nitrogen by metabolizing nitrite to ammonium. The effect of temperature and pH on the establishment of these mutualisms was evaluated and pH was found to be a key determinant for mutualism formation under obligatory conditions. Moreover, the combinations of the two species under non-obligatory growth conditions led to improvement in growth rate and biomass production when compared to single species cultures grown under the same conditions. Such engineered mutualisms are the first step in developing stable multi-species assemblages, while providing a system to generate novel insight into the evolution of mutualistic interactions between phylogenetically distant microorganisms.
- ItemSequence-based analysis of the vitis vinifera L. cv cabernet sauvignon grape must mycobiome in three South African vineyards employing distinct agronomic systems(Frontiers Media, 2015-11) Setati, Mathabatha E.; Jacobson, Daniel; Bauer, FlorianRecent microbiomic research of agricultural habitats has highlighted tremendous microbial biodiversity associated with such ecosystems. Data generated in vineyards have furthermore highlighted significant regional differences in vineyard biodiversity, hinting at the possibility that such differences might be responsible for regional differences in wine style and character, a hypothesis referred to as “microbial terroir.” The current study further contributes to this body of work by comparing the mycobiome associated with South African (SA) Cabernet Sauvignon grapes in three neighboring vineyards that employ different agronomic approaches, and comparing the outcome with similar data sets from Californian vineyards. The aim of this study was to fully characterize the mycobiomes associated with the grapes from these vineyards. The data revealed approximately 10 times more fungal diversity than what is typically retrieved from culture-based studies. The Biodynamic vineyard was found to harbor a more diverse fungal community (H = 2.6) than the conventional (H = 2.1) and integrated (H = 1.8) vineyards. The data show that ascomycota are the most abundant phylum in the three vineyards, with Aureobasidium pullulans and its close relative Kabatiella microsticta being the most dominant fungi. This is the first report to reveal a high incidence of K. microsticta in the grape/wine ecosystem. Different common wine yeast species, such as Metschnikowia pulcherrima and Starmerella bacillaris dominated the mycobiome in the three vineyards. The data show that the filamentous fungi are the most abundant community in grape must although they are not regarded as relevant during wine fermentation. Comparison of metagenomic datasets from the three SA vineyards and previously published data from Californian vineyards revealed only 25% of the fungi in the SA dataset was also present in the Californian dataset, with greater variation evident amongst ubiquitous epiphytic fungi.
- ItemUnderstanding problem fermentations – a review(South African Society for Enology and Viticulture, 2007) Malherbe, S.; Bauer, Florian; Du Toit, M.Despite advances in winemaking technology and improvements in fermentation control, problem alcoholic and malolactic fermentations remain a major oenological concern worldwide. This is due to possible depreciation of product quality and its consequent negative economic impact. Various factors have been identified and studied overv the years, yet the occurrence of fermentation problems persists. The synergistic effect of the various factors amongst each other provides additional challenges for the study of such fermentations. This literature review summarises the most frequently studied causes of problematic alcoholic and malolactic fermentations and in addition provides a summary of established and some potential new analytical technologies to monitor and investigate the phenomenon of stuck and sluggish fermentations.
- ItemThe vineyard yeast microbiome, a mixed model microbial map(PLoS, 2012-12-26) Setati, Mathabatha Evodia; Jacobson, Daniel; Andong, Ursula-Claire; Bauer, FlorianVineyards harbour a wide variety of microorganisms that play a pivotal role in pre- and post-harvest grape quality and will contribute significantly to the final aromatic properties of wine. The aim of the current study was to investigate the spatial distribution of microbial communities within and between individual vineyard management units. For the first time in such a study, we applied the Theory of Sampling (TOS) to sample gapes from adjacent and well established commercial vineyards within the same terroir unit and from several sampling points within each individual vineyard. Cultivation-based and molecular data sets were generated to capture the spatial heterogeneity in microbial populations within and between vineyards and analysed with novel mixed-model networks, which combine sample correlations and microbial community distribution probabilities. The data demonstrate that farming systems have a significant impact on fungal diversity but more importantly that there is significant species heterogeneity between samples in the same vineyard. Cultivation-based methods confirmed that while the same oxidative yeast species dominated in all vineyards, the least treated vineyard displayed significantly higher species richness, including many yeasts with biocontrol potential. The cultivatable yeast population was not fully representative of the more complex populations seen with molecular methods, and only the molecular data allowed discrimination amongst farming practices with multivariate and network analysis methods. Importantly, yeast species distribution is subject to significant intra-vineyard spatial fluctuations and the frequently reported heterogeneity of tank samples of grapes harvested from single vineyards at the same stage of ripeness might therefore, at least in part, be due to the differing microbiota in different sections of the vineyard.
- ItemWine science in the omics era : the impact of systems biology on the future of wine research(SASEV, 2009) Rossouw, D.; Bauer, FlorianIndustrial wine making confronts viticulturalists, wine makers, process engineers and scientists alike with a bewildering array of independent and semi-independent parameters that can in many cases only be optimized by trial and error. Furthermore, as most parameters are outside of individual control, predictability and consistency of the end product remain difficult to achieve. The traditional wine sciences of viticulture and oenology have been accumulating data sets and generating knowledge and know-how that has resulted in a significant optimization of the vine growing and wine making processes. However, much of these processes remain based on empirical and even anecdotal evidence, and only a small part of all the interactions and cause-effect relationships between individual input and output parameters is scientifically well understood. Indeed, the complexity of the process has prevented a deeper understanding of such interactions and causal relationships. New technologies and methods in the biological and chemical sciences, combined with improved tools of multivariate data analysis, open new opportunities to assess the entire vine growing and wine making process from a more holistic perspective. This review outlines the current efforts to use the tools of systems biology in particular to better understand complex industrial processes such as wine making.
- ItemYeast stress response and fermentation efficiency : how to survive the making of wine(South African Society for Enology and Viticulture, 2000) Bauer, Florian; Pretorius, I. S.Fermentation predictability and wine quality are directly dependent on wine yeast attributes that assist in the rapid establishment of numerical dominance in the early phase of wine fermentation, and that determine the ability to conduct an even and efficient fermentation to obtain a desirable alcohol degree. It is therefore not surprising that the primary selection criteria applied to most wine yeast strain development programmes relate to the overall objective of achieving an efficient conversion of grape sugar to alcohol and carbon dioxide, at a controlled rate and without the development of off-flavours. Numerous factors influence the fermentation performance of wine yeast. Following a successful inoculation of grape must with an appropriate starter culture strain, the ability of a wine yeast to adapt to and cope with the hostile environment and stress conditions prevailing in grape juice fermentation are of vital importance to fermentation performance. There is a direct correlation between fermentation efficiency and stress resistance, which refers to the ability of a yeast strain to adapt efficiently to a changing environment and unfavourable growth conditions. Successful yeast cellular adaptation to changes in extracellular parameters during wine fermentation requires the timely perception (sensing) of chemical or physical environmental parameters, followed by accurate transmission of the information to the relevant compartments of the cell. Chemical parameters perceived during wine fermentation include the availability/concentration of certain nutrients (e.g., fermentable sugars, assimilable nitrogen, oxygen, vitamins, minerals, ergosterol and unsaturated fatty acids) and the presence of inhibitory substances (e.g., ethanol, acetic acid, fatty acids, sulfite, phenolic phytoalexins, mycotoxins, bacterial toxins and agrochemical residues). Signals of a physical nature include temperature, pH, agitation and osmotic pressure. The sensing of these environmental signals is carried out by specific receptor proteins, most of them situated on the cellular surface. Once perceived, the information is transmitted by a network of dedicated, interconnected signal transduction pathways to the relevant cellular compartments which implement the adaptive response, a process referred to as "stress response". Intensive research has focused on elucidating the molecular mechanisms involved in stress responses, which are evolutionarily well conserved. Besides furthering our understanding of the fundamental strategies for adaptation to hostile, industrial environments, and the biological resilience of Saccharomyces cerevisiae, the data are of key importance to the future improvement of wine yeast strains. This review describes the different types of stress experienced by wine yeast cells during their life cycles, summarises our current knowledge of some of the most important molecular processes required for the survival of the yeast cell, and highlights the potential benefits for future yeast strain development which can be derived from this research.