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Random mutagenesis and selection of red yeast mutants capable to utilize particular waste substrates
Čačková, Katarína ; Breierová, Emília (oponent) ; Márová, Ivana (vedoucí práce)
Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.
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Controlled biotechnological production of polyhydroxyalkanoates
Šnajdar, Ondřej ; Pekař, Miloslav (oponent) ; Obruča, Stanislav (vedoucí práce)
This diploma thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Cupriavidus necator H16. Goal of this work was preparation, selection and characterization of mutant strains overproducing PHA. Theoretical focuses on the most important PHA, bacteria Cupriavidus necator and mutagenesis techniques. In practical part mutant strains were prepared through physical and chemical mutagenesis. Mutant strains overproducing PHA were selected by cultivation in mineral medium with oil. For further study, 4 mutant strains overproducing PHA were selected. These mutants were biochemically characterized. Specific activities of several intracellular enzymes including enzymes involved in PHA biosynthesis were measured. Resistance of mutants against oxidative stress was measured as well. Mutant strains overproducing PHA revealed higher enzymatic activities of NADPH producing enzymes. Generally, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH partially inhibits TCA cycle and activates accumulation of PHA. Therefore, activities of acetoacetyl-CoA reductase and PHB synthase, enzymes directly involved in PHA synthesis were higher as compared to wild strain as well as molecular weight of produced materials. It can be concluded that biotechnologically perspective mutagens capable of PHA overproduction can be prepared by application of chemical and physical mutagens.
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Use of some molecular techniques to metabolic characterization of industrially significant yeasts
Kostovová, Iveta ; Brázda, Václav (oponent) ; Kráčmar, Stanislav (oponent) ; Márová, Ivana (vedoucí práce)
Carotenoids, ergosterol and fatty acids are valuable compounds used in food, feed and cosmetic industries. Since their conventional sources depend on weather conditions, season, geographical location, and availability of agricultural land, it is necessary to increase its availability for constantly increasing demand. Microbial production of aforementioned compounds exploiting carotenogenic yeast is promising due to their extraordinary capability to simultaneously produce carotenoids, ergosterol and fatty acids. Hereby Doctoral Thesis was aimed to provide the molecular and metabolic characterisation of various carotenogenic yeasts and their potential for industrial applications. For this purpose, the first experimental parts are focused in great detail on yeast species R. mucilaginosa and R. toruloides, their production properties and the influence of nutritional stress and different carbon sources, such as xylose and glycerol. Besides the detailed characterization of their production properties, were these strains also characterized by molecular methods, including sequence analysis of ITS1, ITS2 and D1/2 of the ribosomal operon and analysis of mini and microsatellite sequences M13 and GTG5. R. toruloides is known as an excellent producer of fatty acids and because of it, it recently became a target carotenogenic yeast for the development of tools for its genetic manipulation. In this work were successfully prepared genetically modified clones of R.toruloides bearing over-expressed genes for DGA1 (diacylglycerol acyltransferase) and GPD1 (glycerol-3-phosphate dehydrogenase). Their production of fatty acids was not higher in comparison to the original strain. Therefore, the next part was focused on the preparation of overproducing mutant strains prepared by random mutagenesis. The combination of nitrogen limitation and the carotenoid production inhibitor diphenylamine led to the successful selection of carotenoid overproducing robust mutant strains with resistance to diphenylamine. The last part of the thesis deals with the production properties of lesser known carotenogenic yeast species among order Sporidiobolales and Cystofilobasidales in comparison to relatively well studied carotenogeic yeast species represented by R. toruloides and P. rhodozyma. The best fatty acid-producing strains were S. metaroseus CCY19-6-20 and C. macerans CCY 10-01-02. Moreover, the best carotenoid producer, R.mucilaginosa CCY 19-04-06, naturally produced lycopene in amounts representing more than 80 % of total carotenoids.
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Klonování, charakterisace a exprese mutantů lidské serinracemasy
Nováková, Ilona ; Konvalinka, Jan (vedoucí práce) ; Brynda, Jiří (oponent)
AAbbssttrraakktt Lidská serinracemasa (hSR) je cytosolický enzym využívající pyridoxal-5'-fosfát, který je lokalizován v centrální nervové soustavě. Syntetizuje D-serin, aminokyselinu, jež je endogenním koagonistou N-methyl-D-aspartátových (NMDA) receptorů a hraje tak v mozku klíčovou roli v excitační neurotransmisi. Lidská serinracemasa je tudíž slibným cílem při léčbě neurodegenerativních onemocněních spojených s NMDA receptory. Zatím však bylo identifikováno jen málo specifických inhibitorů a krystalová struktura hSR byla vyřešena teprve nedávno. Rozhodli jsme se tedy pomocí náhodné mutageneze určit aminokyselinové zbytky klíčové pro aktivitu enzymu. O zbytku Ser 84 je známé, že se spolu s Lys 56 podílí na katalýze. Poté, co jsme analyzovali mutanta S84G/P111L lidské serinracemasy, který si zachovával schopnost konvertovat L-serin na pyruvát, jsme syntetizovali a charakterizovali mutantní hSR S84G, abychom mohli vyloučit případný vliv mutace P111L. KKllííččoovváá sslloovvaa:: D-serin; Serinracemasa; Enzymy závislé na PLP; Mutageneze; Racemasy
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Use of some molecular techniques to metabolic characterization of industrially significant yeasts
Kostovová, Iveta ; Brázda, Václav (oponent) ; Kráčmar, Stanislav (oponent) ; Márová, Ivana (vedoucí práce)
Carotenoids, ergosterol and fatty acids are valuable compounds used in food, feed and cosmetic industries. Since their conventional sources depend on weather conditions, season, geographical location, and availability of agricultural land, it is necessary to increase its availability for constantly increasing demand. Microbial production of aforementioned compounds exploiting carotenogenic yeast is promising due to their extraordinary capability to simultaneously produce carotenoids, ergosterol and fatty acids. Hereby Doctoral Thesis was aimed to provide the molecular and metabolic characterisation of various carotenogenic yeasts and their potential for industrial applications. For this purpose, the first experimental parts are focused in great detail on yeast species R. mucilaginosa and R. toruloides, their production properties and the influence of nutritional stress and different carbon sources, such as xylose and glycerol. Besides the detailed characterization of their production properties, were these strains also characterized by molecular methods, including sequence analysis of ITS1, ITS2 and D1/2 of the ribosomal operon and analysis of mini and microsatellite sequences M13 and GTG5. R. toruloides is known as an excellent producer of fatty acids and because of it, it recently became a target carotenogenic yeast for the development of tools for its genetic manipulation. In this work were successfully prepared genetically modified clones of R.toruloides bearing over-expressed genes for DGA1 (diacylglycerol acyltransferase) and GPD1 (glycerol-3-phosphate dehydrogenase). Their production of fatty acids was not higher in comparison to the original strain. Therefore, the next part was focused on the preparation of overproducing mutant strains prepared by random mutagenesis. The combination of nitrogen limitation and the carotenoid production inhibitor diphenylamine led to the successful selection of carotenoid overproducing robust mutant strains with resistance to diphenylamine. The last part of the thesis deals with the production properties of lesser known carotenogenic yeast species among order Sporidiobolales and Cystofilobasidales in comparison to relatively well studied carotenogeic yeast species represented by R. toruloides and P. rhodozyma. The best fatty acid-producing strains were S. metaroseus CCY19-6-20 and C. macerans CCY 10-01-02. Moreover, the best carotenoid producer, R.mucilaginosa CCY 19-04-06, naturally produced lycopene in amounts representing more than 80 % of total carotenoids.
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Klonování, charakterisace a exprese mutantů lidské serinracemasy
Nováková, Ilona ; Brynda, Jiří (oponent) ; Konvalinka, Jan (vedoucí práce)
AAbbssttrraakktt Lidská serinracemasa (hSR) je cytosolický enzym využívající pyridoxal-5'-fosfát, který je lokalizován v centrální nervové soustavě. Syntetizuje D-serin, aminokyselinu, jež je endogenním koagonistou N-methyl-D-aspartátových (NMDA) receptorů a hraje tak v mozku klíčovou roli v excitační neurotransmisi. Lidská serinracemasa je tudíž slibným cílem při léčbě neurodegenerativních onemocněních spojených s NMDA receptory. Zatím však bylo identifikováno jen málo specifických inhibitorů a krystalová struktura hSR byla vyřešena teprve nedávno. Rozhodli jsme se tedy pomocí náhodné mutageneze určit aminokyselinové zbytky klíčové pro aktivitu enzymu. O zbytku Ser 84 je známé, že se spolu s Lys 56 podílí na katalýze. Poté, co jsme analyzovali mutanta S84G/P111L lidské serinracemasy, který si zachovával schopnost konvertovat L-serin na pyruvát, jsme syntetizovali a charakterizovali mutantní hSR S84G, abychom mohli vyloučit případný vliv mutace P111L. KKllííččoovváá sslloovvaa:: D-serin; Serinracemasa; Enzymy závislé na PLP; Mutageneze; Racemasy
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Random mutagenesis and selection of red yeast mutants capable to utilize particular waste substrates
Čačková, Katarína ; Breierová, Emília (oponent) ; Márová, Ivana (vedoucí práce)
Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.
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Controlled biotechnological production of polyhydroxyalkanoates
Šnajdar, Ondřej ; Pekař, Miloslav (oponent) ; Obruča, Stanislav (vedoucí práce)
This diploma thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Cupriavidus necator H16. Goal of this work was preparation, selection and characterization of mutant strains overproducing PHA. Theoretical focuses on the most important PHA, bacteria Cupriavidus necator and mutagenesis techniques. In practical part mutant strains were prepared through physical and chemical mutagenesis. Mutant strains overproducing PHA were selected by cultivation in mineral medium with oil. For further study, 4 mutant strains overproducing PHA were selected. These mutants were biochemically characterized. Specific activities of several intracellular enzymes including enzymes involved in PHA biosynthesis were measured. Resistance of mutants against oxidative stress was measured as well. Mutant strains overproducing PHA revealed higher enzymatic activities of NADPH producing enzymes. Generally, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH partially inhibits TCA cycle and activates accumulation of PHA. Therefore, activities of acetoacetyl-CoA reductase and PHB synthase, enzymes directly involved in PHA synthesis were higher as compared to wild strain as well as molecular weight of produced materials. It can be concluded that biotechnologically perspective mutagens capable of PHA overproduction can be prepared by application of chemical and physical mutagens.
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