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Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Production of pigments by yeasts
Gonová, Dominika ; Hlaváček, Viliam (referee) ; Němcová, Andrea (advisor)
Carotenoids are naturally occurring pigments synthesized by yeast, bacteria, filamentous fungi and plants. In recent years, the interest in the study of these pigments and their microbiological production is increasing mainly due to significant biological effects attributed to carotenoids. This work is conceived as comparative study of six carotenogenic yeasts: two strains of species Rhodotorula glutinis, Rhodotorula mucilaginosa, Sporobolomyces roseus, Sporobolomyces metaroseus a Sporobolomyces pararoseus. Their cultivation was carried out in various media where the oxidative stress was applied in the form of hydrogen peroxide and also waste material – egg pasta – was used as a nutrition source. All studied strains were able to use waste substrate as a source of nutrients, in case of the genus Sporobolomyces increasing production of metabolites was observed. Under the oxidative stress, the majority of the studied yeast showed overproduction of carotenoids and accompanying lipid substances. Sporobolomyces roseus CCY 19-6-4 was characterized by the highest production of carotenoids (1923.78 µg/g -carotene) at the egg pasta media, but on the other hand, it showed a significant decline in biomass. As potentially the best producer of biomass (9.85 g/l) and carotenoids (780.24 µg/g of -carotene), the yeast Rhodotorula glutinis CCY 20-2-26 was observed. Pulcherrimin, synthesized mainly by yeast but also by some spore-forming bacteria, also belongs among natural pigments. Recently, its antagonistic effect that was shown against several microorganisms has been intensively studied, as it seems to be very relevant in regard to biological control. The second part of the work deals with the regulation of pulcherrimin production and its antimicrobial activity. As the pigment-producing yeast strains were used Metschnikowia pulcherrima 145, Metschnikowia pulcherrima 147, Mestchnikowia pulcherrima 149, Metschnikowia andauensis 129, which were cultured on various media containing different concentrations of metal ions and hydrogen peroxide as exogenous stress. In the study of pulcherrimin antagonistic effect, gram-positive bacteria Lactobacillus sakei, gram-negative bacteria Escherichia coli, yeast Candida glabrata and filamentous fungi Phanrochaete chrysosporium were used as tested microorganisms. All studied yeasts were able to produce pulcherrimin, and therefore to show antagonist effect, only in media supplemented with iron, while the color intensity was proportional to the concentration of iron. Antimicrobial activity of pulcherrimin was not observed only against bacteria Escherichia coli.
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Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
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Production of pigments by yeasts
Gonová, Dominika ; Hlaváček, Viliam (referee) ; Němcová, Andrea (advisor)
Carotenoids are naturally occurring pigments synthesized by yeast, bacteria, filamentous fungi and plants. In recent years, the interest in the study of these pigments and their microbiological production is increasing mainly due to significant biological effects attributed to carotenoids. This work is conceived as comparative study of six carotenogenic yeasts: two strains of species Rhodotorula glutinis, Rhodotorula mucilaginosa, Sporobolomyces roseus, Sporobolomyces metaroseus a Sporobolomyces pararoseus. Their cultivation was carried out in various media where the oxidative stress was applied in the form of hydrogen peroxide and also waste material – egg pasta – was used as a nutrition source. All studied strains were able to use waste substrate as a source of nutrients, in case of the genus Sporobolomyces increasing production of metabolites was observed. Under the oxidative stress, the majority of the studied yeast showed overproduction of carotenoids and accompanying lipid substances. Sporobolomyces roseus CCY 19-6-4 was characterized by the highest production of carotenoids (1923.78 µg/g -carotene) at the egg pasta media, but on the other hand, it showed a significant decline in biomass. As potentially the best producer of biomass (9.85 g/l) and carotenoids (780.24 µg/g of -carotene), the yeast Rhodotorula glutinis CCY 20-2-26 was observed. Pulcherrimin, synthesized mainly by yeast but also by some spore-forming bacteria, also belongs among natural pigments. Recently, its antagonistic effect that was shown against several microorganisms has been intensively studied, as it seems to be very relevant in regard to biological control. The second part of the work deals with the regulation of pulcherrimin production and its antimicrobial activity. As the pigment-producing yeast strains were used Metschnikowia pulcherrima 145, Metschnikowia pulcherrima 147, Mestchnikowia pulcherrima 149, Metschnikowia andauensis 129, which were cultured on various media containing different concentrations of metal ions and hydrogen peroxide as exogenous stress. In the study of pulcherrimin antagonistic effect, gram-positive bacteria Lactobacillus sakei, gram-negative bacteria Escherichia coli, yeast Candida glabrata and filamentous fungi Phanrochaete chrysosporium were used as tested microorganisms. All studied yeasts were able to produce pulcherrimin, and therefore to show antagonist effect, only in media supplemented with iron, while the color intensity was proportional to the concentration of iron. Antimicrobial activity of pulcherrimin was not observed only against bacteria Escherichia coli.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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