Department of Viticulture and Oenology

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Analysis of Saccharomyces cerevisiae deletion mutants displaying a modified carbon flux under wine fermentative conditions
(Stellenbosch : University of Stellenbosch, 2009-03) Madlanga, Ncedile Hamilton; Bauer, Florian; Bosch, S.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
Saccharomyces cerevisiae has been used for millennia for the leavening of dough and in the production of alcoholic beverages such as beer and wine. More recently, it is being used as cell factories for the production of important pharmaceutical products. S. cerevisiae has also been extensively used as a model organism for studying many genetic and biochemical processes within the eukaryotic cell. Since the completion of a yeast genome sequence, many functional analysis projects have emerged with the aim of elucidating the functions of the unidentified genes revealed by the genome sequence. One of the most relevant approaches consisted in the construction of a collection of mutants deficient in all single genes, either in a haploid background for non-essential genes, or as heterozygous diploids for essential genes. This collection of strains can be subjected to phenotypic screens that might reveal the function of unknown genes or add to our understanding of already annotated genes. While this approach is promising, it also bears some limitations. For instance, many mutants have no overt phenotypes and some phenotypes do not obviously showcase the function of the encoded protein. In this study, S. cerevisiae strains with single deletions of genes involved in pyruvate metabolism were selected from the Euroscarf deletion library. Pyruvate is a central intermediate of glycolysis, and pyruvate metabolism largely defines the general distribution of carbon flux in the cell. These mutants were screened for modified fermentation kinetics or modified carbon flux under wine fermentative conditions, an environment that had not been previously used for the analysis of these mutants. A strain disrupted in the PDA1 gene, which encodes the E1α subunit of the pyruvate dehydrogenase showed a significant change in phenotype when grown in wine fermentative conditions. In particular, the mutant displayed a prolonged lag phase, but upon entering exponential growth, fermented significantly faster than the wild type strain and completed alcoholic fermentation in a shorter period of time. This phenotype could be of significant industrial interest. The mutant phenotype was further investigated through disruption of the gene in the same as well as in different genetic backgrounds, and through complementation of the PDA1 deletion with a plasmid-born wild type copy. The data show that the PDA1 gene disruption is not solely responsible for the observed phenotypes under wine fermentative conditions. We therefore propose that secondary mutations have contributed to the mutant phenotype. This study shows that phenotypes attributed to a specific gene in mutants of the Euroscarf library should always be confirmed before performing consequent experiments and drawing significant conclusions.
The breeding of yeast strains for novel oenological outcomes
(Stellenbosch : University of Stellenbosch, 2005-12) Mocke, Bernard A; Van Rensburg, P.; Bauer, Florian; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
The quality of wine is influenced by a variety of factors, most noticeably the quality of the grapes, winemaking practices and the yeast strains used for alcoholic fermentation. Although several yeast strains are present in the must at the beginning of fermentation, strains of S. cerevisiae quickly dominate and survive alcoholic fermentations. This dominance of S. cerevisiae prompted research that led to the development of a multitude of industrial yeast starter cultures. Starter cultures are usually capable of quick and complete fermentations, with minimal production of deleterious substances such as volatile acidity, H2S, SO2 and ethyl carbamate. Yeast strains should be able to survive the stressful environment created during alcoholic fermentation, whilst possibly offering novel oenological benefits such as pectinolytic activity, killer activity and malic acid degradation. The increased production of volatile esters and higher alcohols may also be desirable, as this will allow the production of wines that are more aromatic. In this study, VIN13 was crossed with S. paradoxus strain RO88 and WE14 by using a micomanipulator. VIN13 was chosen for its fast and complete fermentation ability and moderate aroma production potential. Other factors such as the presence of killer activity and low production of volatile sulphur compounds also favoured the selection of VIN13. S. paradoxus strain RO88 was selected for its ability to degrade malic acid and the favourable impact on aroma production during fermentation. Hybrids between these yeasts may have the potential to produce more aromatic wines, with the added bonus of pectinolytic activity and a strong fermentation capacity. The first crossing yielded 5 hybrids between VIN13 and S. paradoxus strain RO88. Two of these hybrids stood out in the sense that they were able to degrade more malic acid than VIN13 and they also possessed killer and pectinolytic activity. Cinsaut wine was made and the 2 hybrids were shown to have higher aroma compound capacity than the parental yeasts. This was also confirmed during sensory evaluation. The second crossing between VIN13 and WE14 yielded 10 hybrids with low H2S production potential and killer activity. WE14 was selected for its ability to produce very aromatic wines and also the slower fermentation capacity. Hybrids between these yeast may have the potential to produce wines with an increased aromatic content and the fermentation rate might be slower, thereby improving the aroma profile of the wine. After microvinification, 5 hybrids were selected on the basis of fermentation rate differing from that of the parental yeasts and favourable oenological traits, such as fast and complete fermentation, high production of glycerol and low production of volatile acidity. Pinotage wine was made and it was shown that some of the hybrids produced more esters and higher alcohols than the parental yeasts. Sensory evaluation also showed the aroma production potential of the hybrids, as some of the hybrids were shown to score higher for banana, cherry and tobacco characteristics.
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.
Channelling metabolic flux away from ethanol production by modification of gene expression under wine fermentation conditions
(Stellenbosch : Stellenbosch University, 2013-03) Heyns, Eva Hutton; Bauer, Florian; Setati, Mathabatha Evodia ; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: There is a global demand for technologies to reduce ethanol levels in wine without compromising wine quality. While several chemical and physical methods have been developed to reduce ethanol in finished wine, the target of an industrially applicable biological solution has thus far not been met. Most attempted biological strategies have focused on developing new strains of the main fermentative organism, the yeast Saccharomyces cerevisiae. Gene modification approaches have primarily focused on partially redirecting yeast carbon metabolism away from ethanol production towards glycerol production. These techniques have met with some moderate success, thus the focus of the current study was to re-direct carbon flux towards trehalose production by moderate over-expression of the TPS1 gene. This gene encodes trehalose-6-phosphate synthase, which converts glucose 6-phosphate and UDPglucose to α,α-trehalose 6-phosphate. Previous data have shown that the overproduction of trehalose restricts hexokinase activity reducing the amount of glucose that enters glycolysis. Nevertheless, preliminary TPS1 over-expression studies using multiple copy plasmids have shown some promise, but also indicated significant negative impact on the general fermentation behaviour of strains. In order to reduce such negative impacts of excessive trehalose production, a new strategy consisting in increasing the expression of TPS1 only during specific growth phases and by a relatively minor degree was investigated. Our study employed a lowcopy number episomal vector to drive moderate over-expression of the TPS1 gene in the widely used industrial strain VIN13 at different stages during fermentation. The fermentations were performed in synthetic must with sugar levels representative of those found in real grape must. This, as well as the use of an industrial yeast strain, makes it easier to relate our results to real winemaking conditions. A reduction in fermentation capacity was observed for all transformed strains and controls. Expression profiles suggest that the DUT1 promoter certainly results in increased TPS1 expression (up to 40%) during early exponential growth phase compared to the wild type strain (VIN13). TPS1 expression under the control of the GIP2 promoter region showed increased expression levels during early stationary phase (up to 60%). Chemical analysis of the yeast and the must at the end after fermentation showed an increase in trehalose production =in line with the expression data of TPS1. Importantly, glycerol production was also slightly increased, but without affecting acetic acid levels for the transformed strains. Although ethanol yield is not significantly lower in the DUT1-TPDS1 strain, s statistically significantly lower ethanol yield is observed for over-expression under the GIP2 promotor. Increasing trehalose production during stationary phase appears therefore to be a more promising approach at lowering ethanol yield and redirecting flux away from ethanol production. This controlled, growth phase specific over expression suggests a unique approach of lowering ethanol yield while not impacting on the redox balance.
Characterisation of Wickerhamomyces anomalus and Kazachstania aerobia : investigating fermentation kinetics and aroma production
(Stellenbosch : Stellenbosch University, 2016-12) Lombard, Judith; Bauer, Florian; Musarurwa, Hannibal; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Non-Saccharomyces yeasts have been studied extensively in the past two decades to use as catalysts for adjusting the aroma and chemical properties of wine. Many non-Saccharomyces yeasts dominate in grape must, but Wickerhamomyces anomalus and Kazachstania aerobia have recently been found to be more dominant in several musts in South Africa than what has been reported from other wine growing areas. It has been hypothesised that regional microflora can lead to a terroir specific wine. To further establish these claims, the impact of these non-Saccharomyces yeasts on the chemical profile and sensory perception of wine, in particular when present in high numbers, has yet to be fully elucidated. This study was designed to better characterise isolated strains of non-Saccharomyces species, determining its phenotypic space, as well as to assess their fermentation potential and volatile aroma compound production in synthetic and real grape must. Eight K. aerobia and thirteen W. anomalus isolates were used for characterisation. DNA based taxonomic differences between isolates were investigated using the Random Amplification of Polymorphic DNA (RAPD) method and phenotypic heterogeneity was established using stress assays to determine heat, saline, osmotic and oxidative stress tolerance. Phenotypically diverse K. aerobia and W. anomalus strains were then selected for co- and sequential fermentations with two S. cerevisiae strains, VIN13 and EC1118, in synthetic grape must. In addition, sequential culture fermentations were conducted in Sauvignon blanc grape must by individually pairing two strains of K. aerobia and two strains of W. anomalus with S. cerevisiae EC1118. Wine aroma compounds were quantified using GC-FID. RAPD analysis classified W. anomalus isolates into five distinct groups according to place of origin. Phenotypic variations were evident within and between the proposed strains as was exhibited by heterogeneous resistance to oxidative, saline and osmotic stresses compared to S. cerevisiae, VIN13. The K. aerobia isolates showed no marked genetic differences, although exhibiting slight variations in stress responses. During fermentation the non-Saccharomyces yeasts persisted for longer when S. cerevisiae was only inoculated after 48 hours, or at a lower density. The longer the non-Saccharomyces yeasts proliferated in the must the more pronounced was the effect on aroma production. Kazachstania aerobia yeasts did not achieve a high biomass compared to W. anomalus, but survived for longer in fermentation, especially in Sauvignon blanc grape must. Although W. anomalus displayed strong growth, it was inhibited by the growth of S. cerevisiae. Kazachstania aerobia and W. anomalus gave a unique aroma profile to the wines. The latter yeast produced high concentrations of ethyl acetate, while K. aerobia was characterised by increased acetic acid concentration. Most aroma compounds were increased in mixed culture fermentations, especially higher alcohols, with a significant increase in the esters 2-phenylethyl acetate by K. aerobia, and ethyl caproate and caprylate by W. anomalus. Although, as single cultures these yeasts did not ferment wines to dryness in synthetic grape must and only completed fermentation after 28 days in Sauvignon blanc grape must, they are capable of conferring favourable wine aroma when in association with S. cerevisiae strains with no risk of sluggish fermentation. This study provides a basis for future work on wine quality improvement through exploitation of non-Saccharomyces yeasts and gives insight to the possible impact of K. aerobia and W. anomalus present in grape must in a South African context.
Characterising sensory interactions between volatile phenols and other taint-causing compounds in South African red wines
(Stellenbosch : Stellenbosch University, 2019-04) McKay, Marianne; Buica, Astrid; Bauer, Florian; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
ENGLISH ABSTRACT: South African red wine competes for limited market space, and must maintain consistent, excellent quality. One of the important modalities in assessing the quality and typicality of wine is odour perception (Hopfer et al., 2015). Most investigations quantify aroma compounds by chemical/analytical means and compare levels to odour detection thresholds (ODTs) provided by scientific literature. If malodourous compounds are present at concentrations above their ODTs, they may be considered a threat to wine quality as they exhibit odour activity values >1 (Louw et al., 2010; Prida & Chatonnet, 2010). A review of the literature reveals that studies in wine can use inappropriate ODTs for work carried out in a new wine matrix, and matrix effects on the activity and perception of a compound are often ignored. To properly scientifically evaluate the effect of any compound to wine aroma, formal sensory evaluation in the study matrix is essential (Villamor & Ross, 2013; Perry & Hayes, 2016). Certain volatile phenols (VPs) can contribute to a continuum of smoke-taint related off-flavours including ‘burnt’, ‘bretty’, ‘smoky’, and ‘ashy’ attributes in wine (Jiranek, 2011; Kennison et al., 2011) at higher levels but are generally accepted as being benign to wine aroma at subthreshold levels (Boidron et al., 1988; Prida & Chatonnet, 2010). Useful sensory studies on VPs in wine have been carried out (Simpson et al., 1986; Boidron et al., 1988; Chatonnet et al., 1992; Kennison et al., 2008; Petrozziello et al., 2014), but at supra-threshold levels, and the effect of combinations of subthreshold levels of VPs is not taken into account. The main aims of this research were to characterise the sensory contribution of specific VPs at low levels to red wine odour, and to assess their effect on the perception of attributes through interactions with themselves and other compounds known to be involved in specific wine offflavours. Chapter 2 addresses key knowledge and gaps within the literature around origin, perception and odour detection threshold of VPs associated with specific off-flavours (including ‘smoke’ and ‘ashiness’), and previous studies concerning interaction of aroma compounds which may have relevance to the current study. Chapter 3 investigated trends within the chemical (GC-MS) and sensory (Descriptive Analysis) results for twelve commercial samples of smoke-affected wine. Associations between negative attributes and bushfire events prior to harvest were found. Results also showed that certain sensory effects could have resulted from combinations of subthreshold levels of VPs. The need arose to formally test perceptual interactions at subthreshold levels to see if various effects could be explained. It was thus decided to investigate effects of three VPs: guaiacol, ortho-cresol, 4-ethylphenol, and two compounds associated with certain off-flavours in wine, viz. 3- isobutyl-2- methoxypyrazine (IBMP) and 2,4,6-trichloroanisole (TCA). The work was carried out in partially de-aromatised Shiraz, and it was necessary to establish if ODTs provided in the literature were appropriate for this matrix. Formal sensory work is known to be complex, time-consuming and expensive, particularly in establishing ODTs, and thus a pragmatic sensory approach to the work is outlined in Chapter 4. Chapters 5 and 6 address the perceptual effects of combinations of two, three, four and five offflavour compounds on red wine aroma, which has not been conducted on this scale before. The results of this DA sensory study showed olfactory opposition between clean controls, wines spiked with single compounds (generally fruity and sweet-associated), and wines spiked with complex combinations of VPs and IBMP (linked to negative attributes). Chapter 7 demonstrates another pragmatic approach, using projective mapping (PM) with a large sample size (n=18). Comparable results to the DA interaction study for four compounds in red wine were shown. Chapter 8 investigates effects of combinations of two VPs on four cultivars in order to establish whether there were perceptual olfactory effects that were common to all cultivars, or whether the matrices responded differently from an aroma perception perspective, and shows that samples spiked with combinations of VPs and IBMP show consistently negative olfactory attributes that are independent of cultivar. This research contributes to the sensorial and chemical characterization of selected VPs in red wines, and shows that subthreshold levels of VPs in combination with very low levels of IBMP and TCA can lead to olfactory interactions that cause various olfactory effects, some of them negative. This may help inform winemaking decisions, particularly when dealing with smokeaffected grapes, and/or cultivars that naturally have higher levels of methoxypyrazines, like Merlot and Cabernet Sauvignon. This study also emphasises the importance of understanding effects of VPs on wine aroma, and escalating awareness and sensitivity to these issues in the wine industry.
Characterization of melatonin production and physiological functions in yeast
(Stellenbosch : Stellenbosch University, 2020-03) Motlhalamme, Thato; Bauer, Florian; Prior, Bernard; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
ENGLISH ABSTRACT: Melatonin is a molecule that is found in all living organisms with numerous functions such as the regulation of circadian rhythms in animals and growth stimulation in plants. Studies suggest that melatonin acts as an antioxidant in all living organisms. Its biosynthetic pathways and biological functions are characterised in plants and animals but very little is known about these processes in microorganisms. Yeasts have been found to synthesise melatonin under various conditions such as fermentation, starvation and aerobic growth, but production patterns were found to be inconsistent. The purpose of this study was to investigate the physiological functions of melatonin in yeast in more detail by evaluating a large number of growth conditions to determine conditions that would elicit consistent melatonin production. The study used Saccharomyces cerevisiae as a model organism to study the impact of melatonin on cellular physiology and identify biosynthesis- and melatonin-responsive genes. None of the conditions investigated in this study resulted in consistent melatonin production. When detected, concentrations of melatonin were very low (ng/107 cells) and varied greatly between biological repeats. In plants and animals, melatonin production oscillates in response to diurnal cycles; however, this oscillatory pattern was not observed in the current study. An analysis of the pathway intermediates suggest that multiple enzyme reactions may be involved in the synthesis of melatonin in yeast and that yeast appears not to possess a dedicated synthesis pathway. The absence of orthologs of the enzymes involved in the biosynthetic pathway in yeast supports this conclusion. The chaotic production pattern in yeast suggests that melatonin may be a product of non-specific enzymatic reactions or overflow metabolism. This study took a different approach to evaluating the response of cultures to oxidative stress by conducting experiments in continuous culture conditions instead of batch culture. Over time, the number of differentially expressed genes decreased more rapidly together with yeast recovery from stress in melatonin treated cultures compared to melatonin untreated cultures. Transcriptomic analysis of S. cerevisiae treated with melatonin pre- and post-H2O2 induced oxidative stress suggests that it does not act through any specific stress-responsive pathway and its activity could not be linked to any specific genetic interaction or regulation. However, in the absence of stress, exogenous melatonin enhanced the expression of sulphate assimilation pathway genes. This pathway leads to the formation of methionine and cysteine which are involved in the production of glutathione, and the response therefore may prime cells for subsequent stress. When S. cerevisiae was stressed with various reactive oxygen species generating stressors, melatonin supplementation improved the survival of the cultures in a similar manner to other antioxidants, by increasing the expression of several genes that support the general antioxidant response.
Chitin synthesis in response to environmental stress
(Stellenbosch : Stellenbosch University, 2014-04) Pauw, Marina; Bauer, Florian; Ndlovu, Thulile; Bester, Michael C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Previous studies have indicated that fermentation with yeast strains whose cell walls contain higher chitin levels may lead to reduced wine haze formation. In order to adjust cell wall chitin levels, more information on the regulation of chitin synthesis in wine-relevant yeast is required. Yeast cells are known to increase chitin levels when subjected to certain environmental changes such as an increase in temperature. The main aim of this project was to investigate chitin accumulation and synthesis in wine yeast strains when exposed to environmental change. This was achieved by subjecting the strains to various environmental conditions and comparing chitin levels. The information gained may aid future selection and/or manipulation of yeast strains for the production of higher chitin levels. Three Saccharomyces cerevisiae strains and two Saccharomyces paradoxus strains were subjected to conditions that had been linked to a change in chitin synthesis in past studies in laboratory yeast strains. Of the conditions used in this study, the addition of calcium to a rich media led to the highest cell wall chitin levels. The data also show that chitin synthesis is largely strain dependant. Two conditions which resulted in increased chitin deposition were chosen for gene expression analyses, using strains with strongly diverging average chitin levels. Results showed that an increase in chitin levels correlates with an increase in expression of GFA1, the gene encoding for the first enzyme of the chitin synthesis pathway. Overall, this study provides novel insights into chitin synthesis in Saccharomyces cerevisiae wine yeast strains as well as Saccharomyces paradoxus strains, with possible future implications on haze prevention studies.
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.
Comparative 'omic' profiling of industrial wine yeast strains
(Stellenbosch : Stellenbosch University, 2009-12) Rossouw, D.; Bauer, Florian; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.
Consumer attitudes and sensory perceptions of wine : a South African cross-cultural study
(Stellenbosch : Stellenbosch University, 2018-03) Weightman, Carla Jayne; Nieuwoudt, Helene; Bauer, Florian; Terblanche, Nic; Valentin, Dominique; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The wine consumer landscape has changed in South Africa since 1994. Historically, some population groups had more access and greater cultural affinity for the product “wine”, but such historical patterns are rapidly changing, particularly in urban areas. South Africa has a low per capita wine consumption, but industrial research has shown that there is potential for growth. The wine industry is therefore aiming to become more consumer-driven, in order to increase domestic wine consumption. Consequently, the need for consumer research has increased. Only limited published data is available on South African wine consumers and their consumption habits. The general aim of this study was therefore to do a broad exploration of current wine consumers’ perceptions of wine. This study had a few objectives, firstly to use focus groups to obtain a better understanding of South African wine consumers from different ethnic backgrounds (Black African and Caucasian), with a focus on perceptions, style preferences, context of enjoyment, purchase and consumption patterns, and journeys towards wine consumption. Secondly to develop a questionnaire in order to investigate motivations for wine consumption on a larger scale. Thirdly to compare consumers’, trained assessors, and industry professionals’ sensory perceptions of wine. Lastly to look at the influence of cultivar name on consumer liking. The results of the focus groups showed that the predominant differences occurred between male and female consumers. With regards to ethnicity, this study did not reflect a large distinction between the different ethnic groups. Overall, the wine choice questionnaire showed motivations for drinking wine between the consumer groups to be similar. The social, sensory appeal and ethical concern factors were the three most important motivational factors for South African wine consumers. The industry professionals, trained assessors, and consumers gave very similar answers and distinguished between the sample set of wines in a similar fashion. Consumers that participated in this study mostly described themselves as novices and only somewhat knowledgeable. For them, knowing the cultivar name did not significantly change the way they described the samples, nor did it significantly alter their liking. Even though the samples were all different cultivars made in different styles, on average they were all received positively. This is the first study to focus on understanding the changed landscape of SA wine consumers. The knowledge gained made a significant contribution towards a better understanding of South African consumers’ perceptions of wine, and also highlights directions for future studies.
The control of cellular adhesion of Saccharomyces cerevisiae by the FLO gene regulator Mss11p
(Stellenbosch : University of Stellenbosch, 2010-03) Bester, Michael Christiaan; Bauer, Florian; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour.
A critical process analysis of wine production to improve cost efficiency, wine quality and environmental performance
(Stellenbosch : Stellenbosch University, 2003-03) Sheridan, Craig; Bauer, Florian; Lorenzen, L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Wine cellars are diverse in terms of equipment types and process configurations. Whilst other food production processes have, in many cases, been properly analysed and modelled, this process diversity has resulted in an absence of process analyses in the wine industry. Each wine cellar is unique and represents a fully integrated agro-business, starting with a raw material (grapes) and extending to marketing and selling of the final product (wine). This makes the wine industry unique in this context. This study is the first attempt to analyse winemaking procedures in the form of a process audit. The study was approached in the following manner: • A questionnaire was developed to assess cellar configurations and conditions. This questionnaire was submitted to a statistically significant number of cellars, and a statistically significant number of questionnaires were returned. • The data collected from the questionnaire were statistically analysed and associations between equipment or procedures and wine faults were identified. • Three cellars were studied in depth. These three cellars had their processes audited and their effluent characterised. Additional data were obtained from current sampling projects and these data were analysed to complement the data obtained from the questionnaire • A preliminary input/output model was developed. The major results of this study are: • It was found that certain faults that appear in wine might be associated with equipment and/or process faults. These associations are statistically significant and they show that cellar hygiene is of critical importance when assessing these wine faults. The most important of these faults are VA, microbial contamination of the wine, sluggish and stuck fermentations. A risk hierarchy was derived to indicate which events are associated with others most strongly. • It was found that few wineries measure water consumption and even fewer wineries measure the quantity of effluent produced. • Correlations have been developed to predict winery parameters in terms of tons of grapes pressed per annum. These parameters include water and electricity consumed, wine produced and the quantity of effluent produced. Effluent characteristics have also been correlated to the tons of grapes pressed per annum. These characteristics include chemical oxygen demand, sodium absorption ratio and total dissolved solids in solution. Chemical oxygen demand was identified as the most important contributing factor in winery effluent. It was shown that all variables rise with an increase in cellar size, but the rise is not linear. This implies that large cellars have greater quantities of effluent of lower quality than small cellars. Most cellars have effluent concentrations that require some form of effluent treatment. The characterisation of effluent shows that the most widely used disposal practice is irrigation, and that the effluent disposed in this manner does not meet legislative requirements. • A preliminary input/output model was developed in order to enable wineries that have not measured the relevant parameters to predict the abovementioned variations. The resolution of these predictions is low but the model serves to provide an initial estimate if there are no data available. The model will give industrial averages for any given cellar size. • An economic balance was performed using this preliminary model. It was shown that if cellars were to lower the consumption of utilities and to reduce the strength of their effluent (using cleaner practices and not dilution) the reduction of operating costs could be reduced by 14% for smaller cellars to 17% for larger cellars. This study has shown that it is possible to make wine in a more environmentally friendly manner, producing better quality wines, without incurring extra costs.
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.
The development of yeasts for the optimal production of flavor-active esters and higher alcohols in wine and distillates
(Stellenbosch : Stellenbosch University, 2004-12) Lilly, Mariska; Pretorius, I. S.; Bauer, Florian; Lambrechts, M. G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols during alcoholic fermentation. Some of these esters and higher alcohols are important for the fruity flavors and therefore the final quality of wine and other fermented beverages. Esters are produced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast, ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by the ATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene. Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate, while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fair amount of information is available regarding the ATF 1 gene, limited information is available on the remaining alcohol acetyltransferases. Only two genes that code for esterases have been identified in yeast, namely lAHI and TIPI. It has also been shown that the balance between alcohol acetyltransferases and esterases is important for the net rate of ester accumulation. Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acid metabolic pathway by decarboxylation and reduction. The transamination of the amino acid to the respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain amino acid transferases, which are encoded by the BATI and BAT2 genes, respectively. In recent years, a strong scientific and industrial interest in the metabolism of flavoractive compounds has emerged, but information regarding the roles of specific enzymes and the physiological relevance of their metabolism remains limited. The aim of this project was to investigate the physiological and metabolic consequences of changes in the expression levels of some of the key enzymes involved in aroma compound production. The consequences of these changes on the chemical composition and the fermentation bouquet of wines and distillates were also investigated. The first part of the section on the results in this dissertation reports on the role and relative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism, namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressed in a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeast strain, VIN13. Table wine and base wines for distillation were prepared with these VIN13 transformed strains. The ester concentrations and aroma profiles of the wines and distillates were analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2 increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethyl caproate, while the overexpression of JAHI resulted in a significant decrease in the concentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate, ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease the concentrations of any of the esters. In most cases, there was a correlation between the increase in esters and the decrease in higher alcohols. The data suggest that yeast balances the amount of different esters produced through alcohol acetyltransferases and esterases, and that, in some cases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and the overexpression of two genes, BATl and BAT2, which encode transaminases that contribute to the metabolism of higher alcohols, were investigated. The genes were both disrupted in a S. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wine yeast strain. The effects of these modifications on the general physiology of the corresponding yeast strains and on higher alcohol metabolism were assessed in a range of growth conditions, including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose as sole carbon source and growth in the presence of various concentrations of amino acids. Table wine and base wines for distillation were prepared with the modified industrial strains and the concentrations of the higher alcohols and the aroma profiles of the wine and distillates were analyzed and compared. Batl deletion seemed to be lethal under the conditions that were created, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpression strains, were investigated. These modifications did not appear to significantly affect the general physiology of the strains. The results obtained indicated that the overexpression of BATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesser extent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2 gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid and propionic acid production, and a modest increase in the level of propanol and isovaleric acid. Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamyl acetate concentrations. Sensory analyses indicated that the wines and distillates produced with the strains in which the BATl and BAT2 genes were overexpressed had more fruity characteristics (peach and apricot aromas) than the wines produced by the wild-type strains. This study offers new prospects for the development of wine yeast starter strains with optimized ester and higher alcohol-producing capability that could assist winemakers in their efforts to consistently produce wine to definable specifications and styles and a predetermined flavor profile.
Directed evolution of wine-related lactic acid bacteria and characterisation of evolved strains
(Stellenbosch : Stellenbosch University, 2020-03) Tenyane, Seipati Precious; Bauer, Florian; Du Toit, M.; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
ENGLISH ABSTRACT: Microorganisms form part of complex ecological networks, governed by either metabolic, physical or molecular processes that have positive, neutral or negative effects on microbial interactions. Understanding microbial interactions provides the opportunity to control and manipulate microbes for different biotechnological and industrial applications. For example, the production of beverages such as wine shows how microbial interactions can be controlled and manipulated to achieve desired outcomes. One example is the deliberate inoculation of lactic acid bacteria (LAB) such as Oenococcus oeni or Lactobacillus plantarum to inhibit the growth of spoilage bacteria by depleting available carbon sources such as L-malic acid in a process known as malolactic fermentation (MLF). Indeed, wine provides a good model to study microbial interactions because grape must is inhabited by multiple species of filamentous fungi, yeast, acetic acid bacteria (AAB) and LAB in an anthropogenic and relatively controlled environment. In this study, I investigated the impact of the interaction between the wine yeast Saccharomyces cerevisiae and the LAB L. plantarum. Briefly, the impact of the yeast on the evolution of the bacteria was evaluated after 50 and 100 generations first phenotypically, followed by a genome-wide analysis to identify genetic targets of evolution. A serial transfer method was used for the directed evolution (DE) experiments, introducing bottlenecks and fluctuation between nutrient rich and poor environments after each transfer. This strategy results in a ‘feast-and-famine’ regime, which results in conflicting selective pressures, resembling what normally occurs in dynamic natural environments, which was important here to generate robust and resilient bacteria. Additionally, two yeast strains were used to investigate whether microbial interactions result in yeast-specific adaptations or generic adaptations. Therefore, the yeast strains were kept constant by discarding the yeast at the end of each DE cycle and re-inoculating the mother culture at the start of each DE cycle. The data show yeast strain-specific phenotypes for isolates evolved for 50 generations. Genome-wide analysis showed that broadly targeted pathways are peptidoglycan biosynthesis and degradation, nucleic acid processing, and carbohydrate transport and metabolism in isolates evolved for 50 and 100 generations. These data show that yeast-driven DE results in yeast-specific phenotypic variations and high genetic diversity, but also in convergent evolution over time. The results obtained in this study suggest that yeast drive the evolution of bacteria by dominating the metabolic landscape, showing that strong competitive interactions promote positive selection in mixed species communities, and weak competitive interactions results in no adaptation. This work enriches our understanding of yeast-bacteria interactions over time. Moreover, an isolate that is superior to the parent strain in terms of growth and MLF was obtained, showing potential as a starter culture for winemaking.
Elucidating the metabolic pathways responsible for higher alcohol production in Saccharomyces cerevisiae
(Stellenbosch : University of Stellenbosch, 2011-03) Styger, Gustav; Bauer, Florian; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, 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 genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway.
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 ethanol yields of wine yeast strains under various fermentative conditions
(Stellenbosch : Stellenbosch University, 2014-04) Morgenroth, Olaf; Bauer, Florian; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The market for high quality lower alcohol wines is growing globally. Several factors are responsible for this trend, with socio-economic and health concerns being considered as being the most relevant. It is therefore no surprise that in the past three decades many systems have been developed to reduce wine ethanol levels, each with its own strengths and weaknesses. However, current systems are not always cost effective and frequently result in unwanted side-effects. Microbiological methods primarily based on redirecting carbon flux in existing, or novel Saccharomyces and non-Saccharomyces yeast strains, might have the potential to eliminate or reduce such shortcomings. However, little base-line information regarding differences in ethanol yields of existing wine yeast strains, and on the impact of fermentation conditions on such yields is currently available. In this study the ethanol yield of 15 wine yeast strains was investigated in synthetic wine must under varied wine fermentative conditions including changes in yeast assimilable nitrogen, sugar concentration, pH and fermenting temperatures to identify strains that produce lower ethanol yields and conditions that would favour such an outcome. Most strains and conditions resulted in very similar ethanol yields, however in some cases interesting differences were observed. Some of the strains showed significant differences between high and low nitrogen containing must. Results from synthetic must were confirmed in grape must (Sauvignon Blanc, Chardonnay, Shiraz and Cabernet Sauvignon), but no consistent response could be observed. Interestingly the Shiraz fermentations always showed a higher ethanol yield for all strains investigated. This may be due to a parameter (or combination thereof) which was not included as an experimental factor in our study. Glycerol yield was also studied in the grape must experiments and was found to be more significantly condition dependent than ethanol yield. Temperature and glycerol seemed to be directly proportional confirming the results of previous studies. While temperature did increase glycerol production, it was concluded that the redirection of carbon towards glycerol was not substantial enough to have measurable effect on the final ethanol concentration. The most notable differences which were observed were very specific to a particular yeast strain and condition pairing, thus no generally applicable treatment to achieve lower ethanol yields could be established.

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