Biomedical subjects

Isak S Pretorius

Publications and source records attributed to Isak S Pretorius.

29 records · Page 2Linked to original sources

PubMed2003

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol.

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.

Acyltransferases↗

PubMed2003

The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways.

The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.

Acetyltransferases↗

PubMed2003

Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability.

In Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation. The transcription of MUC1 and of the co-regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p. Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP-PKA pathway. Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals. We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms. We also identify conserved amino acids that are required for the activation function.

Blotting, Northern↗

PubMed2003

Cellular differentiation in response to nutrient availability: The repressor of meiosis, Rme1p, positively regulates invasive growth in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the transition from a nutrient-rich to a nutrient-limited growth medium typically leads to the implementation of a cellular adaptation program that results in invasive growth and/or the formation of pseudohyphae. Complete depletion of essential nutrients, on the other hand, leads either to entry into a nonbudding, metabolically quiescent state referred to as G0 in haploid strains or to meiosis and sporulation in diploids. Entry into meiosis is repressed by the transcriptional regulator Rme1p, a zinc-finger-containing DNA-binding protein. In this article, we show that Rme1p positively regulates invasive growth and starch metabolism in both haploid and diploid strains by directly modifying the transcription of the FLO11 (also known as MUC1) and STA2 genes, which encode a cell wall-associated protein essential for invasive growth and a starch-degrading glucoamylase, respectively. Genetic evidence suggests that Rme1p functions independently of identified signaling modules that regulate invasive growth and of other transcription factors that regulate FLO11 and that the activation of FLO11 is dependent on the presence of a promoter sequence that shows significant homology to identified Rme1p response elements (RREs). The data suggest that Rme1p functions as a central switch between different cellular differentiation pathways.

Base Sequence↗

PubMed2003

Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters.

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.

Acetyltransferases↗

PubMed2003

Flavor-active esters: adding fruitiness to beer.

As they are responsible for the fruity character of fermented beverages, volatile esters constitute an important group of aromatic compounds in beer. In modern high-gravity fermentations, which are performed in tall cylindroconical vessels, the beer ester balance is often sub-optimal, resulting in a clear decrease in beer quality. Despite the intensive research aimed at unravelling the precise mechanism and regulation of ester synthesis, our current knowledge remains far from complete. However, a number of factors that influence flavor-active ester production have already been described, including wort composition, wort aeration and fermentor design. A thoughtful adaptation of these parameters allows brewers to steer ester concentrations and thus to control the fruity character of their beers. This paper reviews the current knowledge of the biochemistry behind yeast ester synthesis and discusses the different factors that allow ester formation to be controlled during brewery fermentation.

Journal Article↗

PubMed2002

Carnitine biosynthesis in Neurospora crassa: identification of a cDNA coding for epsilon-N-trimethyllysine hydroxylase and its functional expression in Saccharomyces cerevisiae.

The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa. The first step of the pathway is catalysed by epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilon-N-trimethyllysine into beta-hydroxy-N-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, Fe2+ and oxygen. Here we report on the cloning of the N. crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae. The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids. The Michaelis-Menten constants of the heterologously expressed enzyme were determined for epsilon-N-trimethyllysine, alpha-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 microM and 46 microM, respectively.

Amino Acid Sequence↗

PubMed2002

Meeting the consumer challenge through genetically customized wine-yeast strains.

Wine producers are facing intensifying competition brought about by a widening gap between wine production and wine consumption, a shift of consumer preferences away from basic commodity wine to top quality wine, and economic globalization. Consequently, they are calling for a total revolution in the 'magical' world of wine. The process of transforming the wine industry from a production- to a market-orientated industry results in an increasing dependence on, amongst others, biotechnological innovation. Market-orientated wine-yeast strains are currently being developed for the cost-competitive production of wine with minimized resource inputs, improved quality and low environmental impact. The emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine.

Consumer Behavior↗

PubMed2002

Genetically tailored grapevines for the wine industry.

Grapevine biotechnology is one of the most promising developments in the global wine industry, which is increasingly faced with conflicting demands from markets, consumers and environmentalists. In the grapevine industries, this technology and its supporting disciplines entail the establishment of stress tolerant and disease resistant varieties of Vitis vinifera, with increased productivity, efficiency, sustainability and environmental friendliness, especially regarding improved pest and disease control, water use efficiency and grape quality. The implementation and successful commercialisation of genetically improved grapevine varieties will only be realized if an array of hurdles, both scientific and otherwise, can be overcome.

Biotechnology↗

PubMed2002

The sensing of nutritional status and the relationship to filamentous growth in Saccharomyces cerevisiae.

Heterotrophic organisms rely on the ingestion of organic molecules or nutrients from the environment to sustain energy and biomass production. Non-motile, unicellular organisms have a limited ability to store nutrients or to take evasive action, and are therefore most directly dependent on the availability of nutrients in their immediate surrounding. Such organisms have evolved numerous developmental options in order to adapt to and to survive the permanently changing nutritional status of the environment. The phenotypical, physiological and molecular nature of nutrient-induced cellular adaptations has been most extensively studied in the yeast Saccharomyces cerevisiae. These studies have revealed a network of sensing mechanisms and of signalling pathways that generate and transmit the information on the nutritional status of the environment to the cellular machinery that implements specific developmental programmes. This review integrates our current knowledge on nutrient sensing and signalling in S. cerevisiae, and suggests how an integrated signalling network may lead to the establishment of a specific developmental programme, namely pseudohyphal differentiation and invasive growth.

Adaptation, Physiological↗

PubMed2002

Microbial cellulose utilization: fundamentals and biotechnology.

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Bacteria↗