Academic literature on the topic 'Saccharomyces cerevisiae – Biotechnology'
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Journal articles on the topic "Saccharomyces cerevisiae – Biotechnology"
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Wimmer, Zdenĕk, Tomás̆ Macek, Ales̆ Svatos̆, and David Šaman. "Bioreductions by Saccharomyces cerevisiae." Journal of Biotechnology 26, no. 2-3 (November 1992): 173–81. http://dx.doi.org/10.1016/0168-1656(92)90005-t.
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THAMMASITTIRONG, SUTTICHA NA-RANONG, THADA CHAMDUANG, UMAPORN PHONROD, and KLANARONG SRIROTH. "Ethanol Production Potential of Ethanol-Tolerant Saccharomyces and Non-Saccharomyces Yeasts." Polish Journal of Microbiology 61, no. 3 (2012): 219–21. http://dx.doi.org/10.33073/pjm-2012-029.
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Four ethanologenic ethanol-tolerant yeast strains, Saccharomyces cerevisiae (ATKU132), Saccharomycodes ludwigii (ATKU47), and Issatchenkia orientalis (ATKU5-60 and ATKU5-70), were isolated by an enrichment technique in yeast extract peptone dextrose (YPD) medium supplemented with 10% (v/v) ethanol at 30°C. Among non-Saccharomyces yeasts, Sd. ludwigii ATKU47 exhibited the highest ethanol-tolerance and ethanol production, which was similar to S. cerevisiae ATKU132. The maximum range of ethanol concentrations produced at 37°C by S. cerevisiae ATKU132 and Sd. ludwigii ATKU47 from an initial D-glucose concentration of 20% (w/v) and 28% (w/v) sugarcane molasses were 9.46-9.82% (w/v) and 8.07-8.32% (w/v), respectively.
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Winans, Matthew J. "Yeast Hybrids in Brewing." Fermentation 8, no. 2 (February 18, 2022): 87. http://dx.doi.org/10.3390/fermentation8020087.
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Microbiology has long been a keystone in fermentation, and innovative yeast molecular biotechnology continues to represent a fruitful frontier in brewing science. Consequently, modern understanding of brewer’s yeast has undergone significant refinement over the last few decades. This publication presents a condensed summation of Saccharomyces species dynamics with an emphasis on the relationship between; traditional Saccharomyces cerevisiae ale yeast, S. pastorianus interspecific hybrids used in lager production, and novel hybrid yeast progress. Moreover, introgression from other Saccharomyces species is briefly addressed. The unique history of Saccharomyces cerevisiae and Saccharomyces hybrids is exemplified by recent genomic sequencing studies aimed at categorizing brewing strains through phylogeny and redefining Saccharomyces species boundaries. Phylogenetic investigations highlight the genomic diversity of Saccharomyces cerevisiae ale strains long known to brewers for their fermentation characteristics and phenotypes. The discovery of genomic contributions from interspecific Saccharomyces species into the genome of S. cerevisiae strains is ever more apparent with increasing research investigating the hybrid nature of modern industrial and historical fermentation yeast.
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Nevoigt, Elke. "Progress in Metabolic Engineering of Saccharomyces cerevisiae." Microbiology and Molecular Biology Reviews 72, no. 3 (September 2008): 379–412. http://dx.doi.org/10.1128/mmbr.00025-07.
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SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.
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Zastrow, C. R., C. Hollatz, P. S. de Araujo, and B. U. Stambuk. "Maltotriose fermentation by Saccharomyces cerevisiae." Journal of Industrial Microbiology and Biotechnology 27, no. 1 (July 1, 2001): 34–38. http://dx.doi.org/10.1038/sj.jim.7000158.
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Lee, Won-Chul, Minho Lee, Jin Woo Jung, Kwang Pyo Kim, and Dongsup Kim. "SCUD: Saccharomyces Cerevisiae Ubiquitination Database." BMC Genomics 9, no. 1 (2008): 440. http://dx.doi.org/10.1186/1471-2164-9-440.
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Iattici, Fabrizio, Martina Catallo, and Lisa Solieri. "Designing New Yeasts for Craft Brewing: When Natural Biodiversity Meets Biotechnology." Beverages 6, no. 1 (January 9, 2020): 3. http://dx.doi.org/10.3390/beverages6010003.
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Beer is a fermented beverage with a history as old as human civilization. Ales and lagers are by far the most common beers; however, diversification is becoming increasingly important in the brewing market and the brewers are continuously interested in improving and extending the range of products, especially in the craft brewery sector. Fermentation is one of the widest spaces for innovation in the brewing process. Besides Saccharomyces cerevisiae ale and Saccharomyces pastorianus lager strains conventionally used in macro-breweries, there is an increasing demand for novel yeast starter cultures tailored for producing beer styles with diversified aroma profiles. Recently, four genetic engineering-free approaches expanded the genetic background and the phenotypic biodiversity of brewing yeasts and allowed novel costumed-designed starter cultures to be developed: (1) the research for new performant S. cerevisiae yeasts from fermented foods alternative to beer; (2) the creation of synthetic hybrids between S. cerevisiae and Saccharomyces non-cerevisiae in order to mimic lager yeasts; (3) the exploitation of evolutionary engineering approaches; (4) the usage of non-Saccharomyces yeasts. Here, we summarized the pro and contra of these approaches and provided an overview on the most recent advances on how brewing yeast genome evolved and domestication took place. The resulting correlation maps between genotypes and relevant brewing phenotypes can assist and further improve the search for novel craft beer starter yeasts, enhancing the portfolio of diversified products offered to the final customer.
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Belloch, Carmela, Roberto Pérez-Torrado, Sara S. González, José E. Pérez-Ortín, José García-Martínez, Amparo Querol, and Eladio Barrio. "Chimeric Genomes of Natural Hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii." Applied and Environmental Microbiology 75, no. 8 (February 27, 2009): 2534–44. http://dx.doi.org/10.1128/aem.02282-08.
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ABSTRACT Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.
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Van der Heggen, Maarten, Sara Martins, Gisela Flores, and Eduardo V. Soares. "Lead toxicity in Saccharomyces cerevisiae." Applied Microbiology and Biotechnology 88, no. 6 (September 1, 2010): 1355–61. http://dx.doi.org/10.1007/s00253-010-2799-5.
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K�tter, Peter, and Michael Ciriacy. "Xylose fermentation by Saccharomyces cerevisiae." Applied Microbiology and Biotechnology 38, no. 6 (March 1993): 776–83. http://dx.doi.org/10.1007/bf00167144.
Full textDissertations / Theses on the topic "Saccharomyces cerevisiae – Biotechnology"
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Leung, Chun Sau. "Modelling studies on a secondary metabolite from Saccharomyces cerevisiae." Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/844537/.
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Modelling studies were performed on a fermentation system using Saccharomyces cerevisiae NCYC 754. The production of fermentation product and cytochrome P-450 were studied under semi-anaerobic condition in batch cultures. The fermentation was carried out in a 5-litre fermenter and controlled at constant set-points which had been optimized by an earlier worker with respect to enzyme yield. An unstructured model was established to describe the biomass profile which comprised two growth phases; however the system did not demonstrate the classical diauxic growth as expected. Furthermore, against the general belief that glucose is the limiting substrate of the system; the maximum wet biomass seemed to depend on the concentration of peptone and yeast extract in the fermentation broth. Growth kinetics indicated that a second substrate was utilized before glucose metabolism began in spite of the presence of high levels of glucose. Luedeking and Piret type models, combined with ethanol inhibition, were derived to describe the profile of ethanol and cytochrome P-450 concentration. Later, it was demonstrated that a close correlation exists between initial glucose and cytochrome P-450 concentration. Viable count by agar plating techniques was used to test the proposed biomass model. The results were in line with the proposed model, even though the cell viability profile in the system was rather low. The Taguchi method was used to seek out the noise factor in the system, and optimize the operating conditions for a particular performance statistic. Contrary to earlier findings, the stirrer speed was found to have little effect on the yield of cytochrome P-450.
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Franken, Jaco. "Carnitine metabolism and biosynthesis in the yeast Saccharomyces cerevisiae." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/4611.
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Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.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.
AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.
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Trollope, Kim. "Investigation of resveratrol production by genetically engineered Saccharomyces cerevisiae strains." Thesis, Stellenbosch : University of Stellenbosch, 2006. http://hdl.handle.net/10019.1/2230.
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Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006.Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.
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Ranwedzi, Ntanganedzeni. "Optimization of β-glucosidase activy in recombinant Saccharomyces cerevisiae strains." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/21731.
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Thesis (MSc)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important quality factors, but these can be influenced by many factors, such as grape-derived compounds that exist as free volatiles and also as glycosidically bound. The chemical composition of wine is determined by factors such as grape variety, geographic position, viticulture condition, microbial ecology of the grape and the winemaking process. The varietals aroma is determined by both the volatile and the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene derivatives, which are naturally present in the wine. Monoterpenes are very important in the flavour and aroma of grapes and wine. They can be found in grapes and wine either in the free, volatile and odorous form, or in the glycosidically-bound, non-volatile and non-odorous form. The ratio of glycosidically-bound compounds to free aroma compounds is very high in the Gewürztraminer, Muscat and Riesling cultivars in particular. The glycosidic bonds can be hydrolysed either by the acid method or by using enzymes. The acid method is disadvantageous because it can modify the monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying the aroma character. The enzyme method of breaking the glycosidic bonds occurs in two successive steps: initial separation of glucose from the terminal sugar by a hydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase, depending on the aglycone moiety), followed by the breaking of the bond between the aglycone and glucose by β-glucosidase. The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera), bacterial, yeast or fungal sources. Most of the enzymes produced by these sources are not functional under the winemaking conditions of low pH, low temperature, high glucose and high ethanol content. However, β-glucosidases from fungal origins, particularly from Aspergillus spp., are tolerant of winemaking conditions. The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii (BGLA), which is linked to the cell wall and the free β-glucosidase, was to determine if anchoring the enzyme to the cell wall will increase the activity of the enzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG 97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmids pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1 anchor domains. All the plasmids were genome-integrated and expressed in the reference strain Saccharomyces cerevisiae 303-1A. All the transformants were grown in 2% cellobiose and showed higher biomass production compared to the reference strain. β-Glucosidase activity was also assayed and transformed strain W16 showed a fourfold increase in activity compared to the reference strain. There was no significant increase in the activity of the other transformed strains, W24, W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was 40ºC. The recombinant strains together with the reference strain were used to make wine from Gewürztraminer grapes. The levels of numerous monoterpenes were enhanced in the resultant wines. The concentration of nerol was increased fourfold, that of citronellol twofold, and geraniol was 20% higher than in the wild type. There was also an increase in the levels of linalool and a-terpinol, but this was not significant. In wines produced with W97, W98 and W24, monoterpene levels did not show a significant difference. In future, the expression of the W16 expression cassette in an industrial wine yeast strain could be performed. In combination with the production of enzymes such as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which are involved in the first step of enzymatic hydrolysis, this wine strain could release the bound monoterpenes and enhance the aroma of the wine.
AFRIKAANSE OPSOMMING: Wyn is ‘n komplekse medium. Wynaroma, -geur en -kleur is belangrike kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan wees of glikosidies gebind is. Die chemiese samestelling van wyn word bepaal deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande, mikrobiese ekologie van die druif en die wynbereidingsproses. Die variëteitsaroma word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene, norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom. Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn. Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in glikosidiesgebinde, nie-vlugtige en nie-geurige vorms. Die verhouding van glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die Gewürztraminer-, Muscat- en Riesling-kultivars. Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode gehidroliseer word. Die nadeel van die suurmetode is dat dit monoterpene kan modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter modifiseer nie. Die ensiemmetode waarmee die glikosidiese verbinding afgebreek word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose van die terminale suiker deur ‘n hidrolase (a-L-arabinofuranosidase, a-Lramnosidase of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur die verbreking van die verbinding tussen die aglikoon en glukose deur β- glukosidase. Die β-glukosidase-ensiem kan vanaf ‘n verskeidenheid plant- (Vitis vinifera), bakterie-, gis- en swambronne verkry word. Die meerderheid van die ensieme wat deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie. β- Glukosidase vanaf ‘n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter wynbereidingstoestande verdra. Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand geanker is. Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie studie gebruik. BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is gekoppel aan CWP2, en in pDLG 98 is dit aan AGa1-ankergebiede gekoppel. Al die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A genoomgeïntegreer en uitgedruk. Al die transformante is in 2% sellobiose gegroei en het hoër biomassaproduksie as die verwysingsras getoon. β-Glukosidaseaktiwiteit is ook geëssaieer en die getransformeerde ras W16 het ‘n viervoudige verhoging in aktiwiteit in vergelyking met die verwysingsras getoon. Daar was geen noemenswaardige verhoging in die aktiwiteit van die ander getransformeerde rasse, W24, W97 en W98, nie. Ensimatiese karakterisering vir optimum-pH en - temperatuur is gedoen – vir al die rasse was die optimum-pH 4 en die optimumtemperatuur 40ºC. Die rekombinante rasse, tesame met die verwysingsras, is gebruik om wyn met Gewürtztraminer-druiwe te maak. Die vlakke van talryke monoterpene is in die gevolglike wyne verhoog. Die konsentrasie van nerol is viervoudig verhoog, dié van sitronellol tweevoudig, en geraniol was 20% hoër as in die wilde tipe. Daar was ook ‘n verhoging in die vlakke van linaloöl en a-terpinol, maar hierdie verhoging was nie noemenswaardig nie. In wyne wat met W97, W98 en W24 gemaak is, het die monoterpeenvlakke nie ‘n noemenswaardige verskil getoon nie. In die toekoms sal die uitdrukking van die W16-uitdrukkingskasset in ‘n industriële wyngisras uitgevoer kan word. In kombinasie met die produksie van ensieme soos a-arabinofuranosidase, a-ramnosidase, β-apiosidase, wat in die eerste stap van ensimatiese hidrolise betrokke is, sal hierdie wyngisras die gebonde monoterpene kan vrylaat en die aroma van die wyn kan verbeter.
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Wilcox, Dale Adrian. "Optimization and evaluation of heterologous lysozyme production in saccharomyces cerevisiae." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/6690.
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Thesis (MSc)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive bacterial cell walls, resulting in cell death. It is thus active against lactic acid bacteria (LAB), which may be present in grape juices and musts. These bacteria are responsible for malolactic fermentation of wines although many species, particularly of the genera Lactobacillus and Pediococcus, are considered spoilage organisms. The growth of LAB is therefore closely monitored and controlled during winemaking. The most common means of control is growth inhibition by chemical treatment (usually with SO2). Lysozyme is a commonly used wine processing aid, complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2 to be used, thus improving the wholesomeness of wine. The OIV (Organisation Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per liter of wine in 1997. This study evaluated the effect of different secretion signals on the secretion of lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate kinase 1) prompter and terminator was also evaluated. In the case of FY23 four secretion signals were used, namely the native lysozyme signal and the S. cerevisiae mating factor-a signal as well as mutants of these signals. These mutants incorporated two additional arginines at the N-terminus of the signals immediately downstream of the terminal methionine. The effect of these mutations was to increase the positive charge of the secretion signal N-terminals. The secretion signal-lysozyme fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter and terminator. The resulting expression cassettes were cloned into integrating vectors YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to transform FY23 and VIN13. FY23 as well as VIN13 transformants were evaluated in an artificial medium designed to reflect the nutrient content of grape juice, with particular attention being paid to assiminable nitrogen. Three hexose concentrations were tested in order to determine the effect thereof on lysozyme secretion titer. Lysozyme secreted under all tested growth conditions was found to be too low for detection by either enzymatic assay or HPLC-FLD. For this reason secreted lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently, lysozyme concentrations in the concentrates was determined by means of the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was also performed. No significant differences were found between native or MF-a secretion signals and their mutated counterparts in terms of secretion titer or proteolytic maturation. Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases, with both an authentically processed and a larger form, in which the secretion signal was not completely removed, being present. Lysozyme secreted with the native signal appeared to be correctly processed in all cases. Secretion titer from high copy number episomal FY23 tranformants was similar to that of integrants containing a single copy of the gene. Sugar concentration affected lysozyme production, with higher quantities of the enzyme being secreted when higher initial sugar concentrations were used. Lysozyme titers were extremely low (< 0:25 mg/L) with all expression cassettes under all the tested conditions with both FY23 and VIN13. In the case of the VIN13’s a lower final biomass was found for the secretor strain tested in comparrison to the VIN13 wild-type.
AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë (MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling (gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn. Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23 is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF- a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale metionien. Die effek van hierdie mutasies was om die positiewe lading van die uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2, gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees. Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.
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Wordon, Brett Arthur. "The use of fluorescent flow cytometry to evaluate the inactivation of Saccharomyces cerevisiae by sequential application of ultrsound (20kHz) and heat." Thesis, Cape Peninsula University of Technology, 2009. http://hdl.handle.net/20.500.11838/828.
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Thesis (MTech (Food Technology)--Cape Peninsula University of Technology, 2009The primary aim of this study was to establish the effects of both cavitation, (20 KHZ), and heat (55°C or 60•C) on Saccharomyces cerevisiae GC210 (MATa lys2) suspended in physiological saline. Fluorescent flow cytometry was used to determine the effects of moist heat and acoustic cavitation on S. cerevisiae cells. Results from this study could be used as a guide for use by the food industry for the combined use of heat and sonication to disinfect various solutions contaminated with S. cerevisiae.
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De, Villiers Gillian K. "Development of recombinant Saccharomyces cerevisiae for improved D-xylose utilisation." Thesis, Stellenbosch : University of Stellenbosch, 2006. http://hdl.handle.net/10019.1/17346.
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Thesis (MSc)--University of Stellenbosch, 2006.ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol.
AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.
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Louw, Campbell Trout. "Transcriptional regulation of the endo-polygalacturonase-encoding gene in Saccharomyces cerevisiae." Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4005.
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Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities.
AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W) geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1 geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie van die geen sodoende in sommige rasse voorkom. Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens, asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk. Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van wyngiste te verbeter. Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme van regulering van toepassing op PGU1 te identifiseer. As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.
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Kriel, Johan Hendrik. "Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53364.
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Thesis (MSc)--Stellenbosch University, 2003.ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity.
AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
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Jain, Vishist Kumar. "Modifying redox potential and its impact on metabolic fluxes in Saccharomyces cerevisiae." Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/3983.
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Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: The production of glycerol by Saccharomyces cerevisiae under anaerobic conditions is essential for maintaining the intracellular redox balance thereby allowing continuous energy generation through conversion of sugars into ethanol. In addition, glycerol can act as an osmolyte and is synthesized to maintain turgor pressure under hyperosmotic conditions. The production of ethanol from sugars can be a redox-neutral process, where the NAD+ (nicotinamide adenine dinucleotide) that is consumed during the glycolytic conversion of glyceraldehyde-3-phosphate to pyruvate is later regenerated by the reduction of acetaldehyde to ethanol. However, in particular the redirection of metabolic flux of pyruvate to biomass formation leads to excess NADH formation. The intracellular redox balance in these conditions is then primarily maintained through formation of glycerol which is control by two main enzymes, namely Gpd1p and Gpd2p. Deletion of the genes coding for these two proteins leads to accumulation of NADH and renders the cells incapable of maintaining their fermentative ability and growth under anaerobic conditions. The goal of this study was to investigate the growth, fermentative ability and metabolite synthesis of various gpd1Δgpd2Δ double mutant (DM) strains in which the redox balancing potential was partially restored through expression of native or heterologous genes. Strains were constructed by introducing alternative NADH oxidizing pathways or manipulating existing pathways to favour the oxidation of excess NADH. More specifically, the modifications included (i) sorbitol formation; (ii) establishing a pathway for propane-1,2-diol formation; and (iii) increasing ethanol formation. Apart from genetically manipulating the gpd1Δgpd2Δ double mutant, the addition of pyruvate during growth was also investigated. The experiments were carried out under oxygen limited conditions in a high sugar medium and the fermented product was analyzed for total sugar consumed, biomass and primary and secondary metabolites formed by the different strains. The relationships between sugar consumption, growth and metabolite production by different strains were investigated by comparing the data generated from the different strains by using multivariate data analysis tools. Analysis of the pathways involved in the production of primary (acids, ethanol and other metabolites) and secondary metabolites (aroma compounds) were also carried out in order to establish flux modification in comparison to the wild type (WT) strain. The results revealed that these manipulations improved the fermentative capacity of the gpd1Δgpd2Δ double mutant, suggesting a partial recovery of NAD+ regeneration ability, albeit not to the extent of the WT strain. As expected a significant correlation was found between sugar consumption and ethanol and biomass formation. Ethanol yields but not final concentrations were increased by the genetic manipulations. Sorbitol by DM(srlD) and DM(SOR1) strains and propane-1,2-diol by DM(gldA, GRE3, mgsA) strain were formed in significant amounts although at lower molar yields than glycerol. Furthermore, by genetic manipulation the yield of secondary metabolites (isobutanol, isoamyl alcohol, 2-phenyl ethanol and isobutyric acid) was increased whereas the ethyl acetate concentration and yield decreased. The results indicate that aroma compound properties of wine yeasts could be favourably changed by manipulating the glycerol synthesizing pathway. The addition of pyruvate during the growth of gpd1Δgpd2Δ double mutant contributes to excess NADH re-oxidation through additional ethanol formation.
AFRIKAANSE OPSOMMING: Die produksie van gliserol deur Saccharomyces cerevisiae onder anaërobiese toestande is noodsaaklik vir die onderhouding van die intrasellulêre redoksbalans en maak dus ononderbroke energie-ontwikkeling tydens die omsetting van suikers in etanol moontlik. Daarbenewens kan gliserol as ‘n osmoliet optree en word dit gesintetiseer om turgordruk onder hiperosmotiese toestande te onderhou. Die produksie van etanol uit suikers kan ‘n redoksneutrale proses wees, waar die NAD+ (nikotinamiedadenien-dinukleotied) wat tydens die glikolitiese omskakeling van gliseraldehied-3-fosfaat na piruvaat verbruik word, later deur die reduksie van asetaldehied na etanol regenereer word. Die nasending van die metaboliese vloeiing van piruvaat na biomassavorming lei egter na ‘n oormaat NADH-vorming. Onder hierdie toestande word die intrasellulêre redoksbalans dan hoofsaaklik deur die vorming van gliserol onderhou. Laasgenoemde word veral deur twee ensieme beheer, naamlik Gpd1p en Gpd2p. Die delesie van die gene wat vir hierdie twee proteïene enkodeer, lei tot ‘n akkumulasie van NADH en veroorsaak dat die selle nie hulle gistingsvermoë en groei onder anaërobiese toestande kan onderhou nie. Die doelwit van hierdie studie was om die groei, gistingsvermoë en metabolietsintese van verskeie gpd1Δgpd2Δ dubbelmutant (DM) rasse te ondersoek waarin die redoksbalanseringspotensiaal gedeeltelik herstel is deur die uitdrukking van inheemse of heteroloë gene. Rasse is gekonstrueer deur alternatiewe NADH-oksiderende weë in te voer of deur bestaande weë te manipuleer om die oksidasie van oormaat NADH te bevoordeel. Meer spesifiek het die modifikasies die volgende ingesluit: (i) sorbitolvorming; (ii) die vestiging van ‘n weg vir propaan-1,2-diol-vorming; en (iii) die verhoging van etanolvorming. Buiten die genetiese manipulering van die gpd1Δgpd2Δ dubbelmutant, is die byvoeging van piruvaat tydens groei ook ondersoek. Die eksperimente is onder suurstofbeperkte toestande in ‘n hoë-suiker medium uitgevoer en die gegiste produk is ondersoek vir totale suikerverbruik, biomassa en primêre en sekondêre metaboliete wat deur die verskillende rasse gevorm is. Die verhoudings tussen suikerverbruik, groei en metabolietproduksie deur die verskillende rasse is ondersoek deur die data wat deur die verskillende rasse gegeneer is deur middel van meerveranderlike data-analise te vergelyk. Analise van die weë wat in die produksie van primêre (sure, etanol en ander metaboliete) en sekondêre metaboliete (aromaverbindings) betrokke is, is ook uitgevoer om die verandering in vloei te bepaal in vergelyking met die wildetipe (WT) ras. Die resultate het gewys dat hierdie manipulasies die gistingsvermoë van die gpd1Δgpd2Δ-dubbelmutant verbeter het, wat ‘n gedeeltelike herstel van NAD+- regenerasievermoë voorstel, hoewel nie tot dieselfde mate as in die WT-ras nie. Soos verwag, is ‘n beduidende korrelasie tussen suikerverbruik en etanol- en biomassavorming gevind. Etanolopbrengs is deur genetiese manipulasies verhoog, maar nie die finale konsentrasies van etanol nie. Sorbitol is in beduidende hoeveelhede deur die DM(srlD) en DM(SOR1)-rasse gevorm en propaan-1,2-diol deur die DM(gldA, GRE3, mgsA) -rasse, hoewel teen laer molare opbrengste as gliserol. Verder is die opbrengs van sekondêre metaboliete (isobutanol, iso-amielalkohol, 2-fenieletanol en isobottersuur) deur genetiese manipulasie verhoog, terwyl die etielasetaatkonsentrasie en -opbreng verlaag is. Die resultate dui aan dat die aromaverbindingseienskappe van wyngiste voordelig verander kan word deur die gliserolsintetiseringsweg te manipuleer. Die byvoeging van piruvaat tydens die groei van die gpd1Δgpd2Δ-dubbelmutant dra by tot uitermate NADH-reoksidasie tydens die bykomende vorming van etanol.
Books on the topic "Saccharomyces cerevisiae – Biotechnology"
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F, Walton E., and Yarranton G. T, eds. Molecular and cell biology of yeasts. Glasgow: Blackie, 1989.
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Investigations in yeast functional genomics and molecular biology. Toronto: Apple Academic Press, 2014.
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Yeast biotechnology. Wimborne, Dorset: Intercept, 1988.
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Yeast Biotechnology. Springer, 2011.
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R, Berry David, Russell Inge 1947-, and Stewart Graham G. 1942-, eds. Yeast biotechnology. London: Allen & Unwin, 1987.
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Berry, David R. Yeast Biotechnology. Springer, 2011.
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Berry, David R., I. Russell, and G. C. Stewart. Yeast Biotechnology. Springer London, Limited, 2012.
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1934-, Zimmermann F. K., and Entian K. -D, eds. Yeast sugar metabolism: Biochemistry, genetics, biotechnology, and applications. Lancaster, PA: Technomic Pub., 1997.
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(Editor), E. F. Walton, and G. T. Yarranton (Editor), eds. Molecular And Cell Biology Of Yeasts. Springer, 1990.
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F, Walton E., and Yarranton G. T, eds. Molecular and cell biology of yeasts. Blackie, 1989.
Find full textBook chapters on the topic "Saccharomyces cerevisiae – Biotechnology"
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Bon, Elba P. S., Elvira Carvajal, Mike Stanbrough, Donald Rowen, and Boris Magasanik. "Asparaginase II of Saccharomyces cerevisiae." In Biotechnology for Fuels and Chemicals, 203–12. Totowa, NJ: Humana Press, 1997. http://dx.doi.org/10.1007/978-1-4612-2312-2_19.
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Siewers, Verena, Uffe H. Mortensen, and Jens Nielsen. "Genetic Engineering Tools for Saccharomyces cerevisiae." In Manual of Industrial Microbiology and Biotechnology, 287–301. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555816827.ch20.
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Wolf, K., and B. Schäfer. "Mitochondrial Genetics of the Budding Yeast Saccharomyces cerevisiae." In Genetics and Biotechnology, 71–93. Berlin, Heidelberg: Springer Berlin Heidelberg, 2004. http://dx.doi.org/10.1007/978-3-662-07426-8_5.
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Oliveira, Edna M. M., Elvira Carvajal, and Elba P. S. Bon. "L-Asparaginase II of Saccharomyces cerevisiae." In Twentieth Symposium on Biotechnology for Fuels and Chemicals, 311–16. Totowa, NJ: Humana Press, 1999. http://dx.doi.org/10.1007/978-1-4612-1604-9_28.
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Silveira, Maria Cristina F., Edna M. M. Oliveira, Elvira Carvajal, and Elba P. S. Bon. "Nitrogen Regulation of Saccharomyces cerevisiae Invertase." In Twenty-First Symposium on Biotechnology for Fuels and Chemicals, 247–54. Totowa, NJ: Humana Press, 2000. http://dx.doi.org/10.1007/978-1-4612-1392-5_18.
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Hahn-Hägerdal, Bärbel, C. Fredrik Wahlbom, Márk Gárdonyi, Willem H. van Zyl, Ricardo R. Cordero Otero, and Leif J. Jönsson. "Metabolic Engineering of Saccharomyces cerevisiae for Xylose Utilization." In Advances in Biochemical Engineering/Biotechnology, 53–84. Berlin, Heidelberg: Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/3-540-45300-8_4.
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Iwahashi, H., S. Nwaka, K. Obuchi, and Y. Komatsu. "Neutral Trehalases Contribute to Barotolerance in Saccharomyces cerevisiae." In Advances in High Pressure Bioscience and Biotechnology, 69–72. Berlin, Heidelberg: Springer Berlin Heidelberg, 1999. http://dx.doi.org/10.1007/978-3-642-60196-5_15.
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Bravim, Fernanda, Melina Campagnaro Farias, Oeber De Freitas Quadros, and Patricia Machado Bueno Fernandes. "Genetic Enhancement of Saccharomyces Cerevisiae for First and Second Generation Ethanol Production." In Industrial Biotechnology, 239–79. Toronto ; [Hackensack?] New Jersey : Apple Academic Press, 2016.: Apple Academic Press, 2017. http://dx.doi.org/10.1201/9781315366562-10.
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Baumann, Leonie, Florian Wernig, Sandra Born, and Mislav Oreb. "14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives." In Genetics and Biotechnology, 339–68. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-49924-2_14.
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Dhavale, Abhinandan, Atul Vhanmarathi, Shrinivas Deshmukh, and Seema Dabeer. "Unstructured Kinetic Modeling of Glutathione Production by Saccharomyces cerevisiae NCIM 3345." In Biotechnology and Biochemical Engineering, 11–20. Singapore: Springer Singapore, 2016. http://dx.doi.org/10.1007/978-981-10-1920-3_2.
Full textConference papers on the topic "Saccharomyces cerevisiae – Biotechnology"
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Istiqomah, Lusty, Ema Damayanti, Doni Arisnandhy, Francis Maria Constance Sigit Setyabudi, and Muslih Anwar. "Saccharomyces cerevisiae B18 as antifungal and aflatoxin binder in vitro." In 1ST INTERNATIONAL CONFERENCE ON BIOINFORMATICS, BIOTECHNOLOGY, AND BIOMEDICAL ENGINEERING (BIOMIC 2018). Author(s), 2019. http://dx.doi.org/10.1063/1.5098414.
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Cai, Lu, Zhiyong Pei, Sheng Qin, and Xiujuan Zhao. "Prediction of Protein-Protein Interactions in Saccharomyces cerevisiae Based on Protein Secondary Structure." In 2012 International Conference on Biomedical Engineering and Biotechnology (iCBEB). IEEE, 2012. http://dx.doi.org/10.1109/icbeb.2012.302.
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Potoroko, I., A. Paymulina, D. Uskova, N. Popova, and M. Velyamov. "Improvement of Saccharomyces cerevisiae and Lactococcus delbrueckii ssp. bulgaricus bioactivity by sonochemically microstructured fucoidan." In INTERNATIONAL CONFERENCE ON FOOD SCIENCE AND BIOTECHNOLOGY (FSAB 2021). AIP Publishing, 2021. http://dx.doi.org/10.1063/5.0068773.
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Shomrina, I. A., Yu N. Gulyaeva, and V. B. Tishin. "Generalized mathematical kinetic model of Saccharomyces cerevisiae yeast growth in oxygen deficiency." In ACTUAL PROBLEMS OF ORGANIC CHEMISTRY AND BIOTECHNOLOGY (OCBT2020): Proceedings of the International Scientific Conference. AIP Publishing, 2022. http://dx.doi.org/10.1063/5.0069155.
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Wolski, Paul, Alberto Lopes, Kai Deng, Blake Simmons, Aindrila Mukhopadhyay, Steven Singer, and Kenneth Sale. "High Throughput expression and characterization of laccases in Saccharomyces cerevisiae." In Proposed for presentation at the Society for Industrial Microbiology and Biotechnology Symposium on Biomaterials, Fuels, and Chemicals held April 26-28, 2021 in virtual, virtual, US. US DOE, 2021. http://dx.doi.org/10.2172/1866049.
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Wolski, Paul, Alberto Lopes, Kai Deng, Blake Simmons, Aindrila Mukhopadhyay, Steven Singer, and Kenneth Sale. "High Throughput expression and characterization of laccases in Saccharomyces cerevisiae." In Proposed for presentation at the Society for Industrial Microbiology and Biotechnology Symposium on Biomaterials, Fuels, and Chemicals held April 26-28, 2021 in virtual, virtual, US. US DOE, 2021. http://dx.doi.org/10.2172/1866050.
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