Microbial, Biochemical and Food Biotechnology
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Browsing Microbial, Biochemical and Food Biotechnology by Subject "3-Hydroxy oxylipins"
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Item Open Access The lipid composition of the yeast genus Saccharomycopsis Schiönning(University of the Free State, 2004-11) Sebolai, Olihile Moses; Kock, J. L. F.; Smit, M. S.; Van Wyk, P. W. J.English: In this study, the construction of a forecasting model, using intracellular fatty acid composition as indicator, was attempted to assist in the search for yeasts capable of producing 3-hydroxy oxylipins. In order to achieve this, it was first attempted to establish a database mapping the distribution of fatty acids (FAs) associated with the neutral-, glyco- and phospholipid fractions of the 10 species representing the genus Saccharomycopsis. It was possible to identify nine of the 10 species i.e. Saccharomycopsis capsularis, S. crataegensis, S. fibuligera, S. javanensis, S. malanga, S. schoenii, S. selenospora, S. synnaedendra and S vini with the exception of S. fermentans. Saccharomycopsis crataegensis was unique since it produced by far the highest percentage neutral lipids (52.4% w/w) while S. schoenii produced the highest percentage phospholipids (35.9% w/w). All strains produced palmitic- (16:0), stearic- (18:0), oleic- (18:1) and linoleic acid (18:2) in all lipid fractions analysed. The major FAs produced were 18:1 and 18:2, while palmitoleic- (16:1) and a-linolenic acid [18:3 (w-3)] varied between species. Saccharomycopsis capsularis produced the highest percentage 18:2 in the neutral lipid fraction while S. crataegensis, S. malanga and S. selenospora produced the highest percentages of 18:1, 18:0 and, 18:3 (w-3) respectively, in the neutral lipids. Saccharomycopsis vini produced the lowest percentage 16:0 in this fraction. Saccharomycopsis fibuligera and S. schoenii produced the highest percentages of 16:0 and 18:2 respectively in the glycolipid fraction. Saccharomycopsis javanensis and S. synnaedendra produced the highest percentages of 18:1 and 16:1 respectively in the phospholipid fraction. Although it was possible to differentiate between most species using this phenotypic character, these FAs could not be used to predict what kind of 3-OH oxylipins these species are capable of producing. Saccharomycopsis fermentans (novel unidentified 3-OH oxylipin), S. malanga (3-OH 16:0), S. synnaedendra (3-OH 16:0, 3-OH 17:0, 3-OH 18:0, 3-OH 18:1, 3-OH 19:0, 3-OH 19:1, 3-OH 20:0, 3-OH 22:0) and S. vini (3-OH 9:1, 3-OH 10:1) could be separated using this character. Although, S. capsularis and S. javanensis both produced 3-OH 9:1, fatty acids with uneven carbon atoms which may serve as precursors could not be detected in the neutral-, glyco- or phospho-lipid fractions.Item Open Access Oxylipin distribution in Eremothecium(University of the Free State, 2006-11) Leeuw, Ntsoaki Joyce; Kock, J. L. F.; Pohl, C. H.; Van Wyk, P. W. J.English: In the early 1990’s, Kock and co-workers discovered acetylsalicylic acid (ASA)-sensitive oxylipins in yeasts. It was also reported that the site of production of these compounds may serve as important targets to control fungal infections. In 2004, researchers exposed another function for these oxylipins – they may act as lubricants during spore release from enclosed asci. Since oxylipin production in only a limited number of species representing Eremothecium was thus far studied, it became the aim of this project to further extend this study and to determine the type and distribution of 3- hydroxy (OH) oxylipins in the remaining species i.e. Eremothecium coryli, E. cymbalariae and E. gossypii. In addition, the possible functions of these oxylipins as well as ascospore shape and ornamentations were assessed. Finally, the antifungal activity of ASA was also investigated in this group of important plant pathogens as well as other yeasts. Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. In this study, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluoresence confocal laser scanning microscopy it was found that hydrophobic 3-OH oxylipins cover the surfaces of these ascospores. Using gas chromatography-mass spectrometry, only the oxylipin 3-OH 9:1 (a monounsaturated fatty acid consisting of a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies, it was possible to map the presence of these compounds also in other Eremothecium species. In E. cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in E. gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as target for ASA antifungal action.Item Open Access The role of lipids in the flocculation of Saccharomyces cerevisiae(University of the Free State, 2005-11) Strauss, Catharina J; Kock, J. L. F.; Van Wyk, P. W. J.; Lodolo, E. J.English: Although beer production is one of the oldest biotechnologies in the world, a major constraint in brewing remains controlling flocculation. Evidence points towards a possible role of lipids, associated with the cell surfaces, as a major factor responsible for flocculation. Therefore, the aim in this study became to evaluate the contribution of lipids, especially oxylipins, in the flocculation of Saccharomyces cerevisiae UOFS Y-2330. Saccharomyces cerevisiae UOFS Y-2330 was selected as a model, since it was found to demonstrate both Flo1 and NewFlo phenotype flocculation behaviour, when cultivated in different media. In a defined medium with glucose as a sole carbon source, this strain immediately flocculated strongly and lost this ability before stationary phase was reached. In a complex medium containing glucose, this yeast strongly flocculated towards the stationary growth phase without losing this ability during this phase. This inverse pattern may be ascribed to a switch in sensitivity of the yeast to flocculate in the presence of glucose as well as pH level, which may, in turn, influence the availability of calcium ions. In both media, matured cells produced protuberances upon flocculation as observed by electron and immunofluorescence microscopy, which may be involved in cell adhesion. This was followed by further investigations into the role of lipids over the growth cycle of this yeast. Here, it was uncovered that Sacch. cerevisiae UOFS Y-2330 does not only demonstrate inverse flocculation, but is also characterised by two different lipid turnover patterns. During Flo1 phenotype flocculation, this yeast showed two neutral lipid accumulating stages (i.e. at 8 h and from 12 h). This is probably triggered by flocculation, which may be regarded as a survival mechanism where cells accumulate especially neutral lipids as reserve energy source - a similar mechanism is probably operative when cells enter stationary growth. Contrary to Flo1 behaviour, this strain in NewFlo phenotype mode demonstrates only a single lipid accumulation phase i.e. when cells enter stationary growth, which coincides with the increase in flocculation. In addition, an increase in phospholipids was experienced during active growth in both flocculation behaviours, probably as a result of active membrane production. These results prompted us to investigate the possible role of oxylipins present on the cell surfaces during the flocculation process. It was found that some strains of Sacch. cerevisiae (include strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When aspirin was added to cultures of Sacch. cerevisiae UOFS Y-2330, flocculation was significantly inhibited as well as the production of 3-hydroxy (OH) 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3-OH 8:0 could be detected before flocculation onset in this yeast. Next, the involvement of these oxylipins in co-flocculation was assessed. According to the lectin-theory, the yeast Schizosaccharomyces pombe lacks the specific receptors necessary to facilitate co-flocculation with Sacch. cerevisiae species. In this study we demonstrate oxylipin associated co-flocculation between Sacch. cerevisiae UOFS Y-2330 and S. pombe strains using differential cell staining, immunofluorescence and ultrastructural studies. Using a 3-OH oxylipin specific antibody coupled to a fluorescing compound, 3-OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3-OH oxylipins was confirmed using gas chromatography-mass spectrometry. Whether these 3- OH oxylipins play a role in affecting co-flocculation of Sacch. cerevisiae with S. pombe cells through possibly entropic-based hydrophobic interactions and/or hydrogen bonds still needs to be verified. Studies on the physiological, genetic as well as colloidal aspects of flocculation using this model strain may lead to important new insights in this fascinating phenomenon as well as applications in industry.