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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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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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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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