Sprememba fenotipa asimilacije sladkorjev v lokalnih izolatih kvasovk z usmerjeno evolucijo

Ružič, Katja (avtor), Petković, Hrvoje (mentor), Tome, Miha (komentor)

Za uporabo lokalnih izolatov kvasovk v pivovarski industriji je značilna njihova slabša asimilacija alternativnih sladkorjev, kar neposredno vpliva na učinkovitost in hitrost fermentacije v procesu proizvodnje piva. Maltoza, ki se običajno pri kvasovkah v prisotnosti glukoze slabo asimilira zaradi katabolne represije, na koncu fermentacije ostane pretežno neporabljena. Namen tega magistrskega dela je bil z uporabo usmerjene evolucije in mutageneze pripraviti mutante lokalnih izolatov, ki bodo odporne na 2-deoksiglukozo (2-DOG). 2-DOG je antimetabolit glukoze, ki vpliva na glikolizo, ter s tem posredno prisili celico, da aktivira sisteme za porabo alternativnih virov sladkorjev. Glavni cilj magistrskega dela je odpraviti katabolno represijo z glukozo pri dveh izolatih kvasovk vrste Saccharomyces cerevisiae in S. paradoxus in/ali izboljšati privzem maltoze. V prvem delu naloge smo za oba seva določili minimalne inhibitorne koncentracije za 2-DOG na definiranem gojišču z maltozo ter krivulje preživetja pri izpostavitvi mutagenu. V drugem delu je sledila usmerjena evolucija z mutagenezo, s katero smo pridobili mutante odporne na 2-DOG oz. kolonije, ki so kazale hitrejšo rast pri višjih koncentracijah 2-DOG, kot na kontrolnih ploščah brez mutagena. Izbrane mutante smo testirali in s pomočjo HPLC analize določili hitrost porabe glukoze, maltoze in produkcijo etanola ob izbranih časovnih točkah. Ugotovili smo, da smo pri določenih mutantih S. cerevisiae uspeli odpraviti katabolno represijo z glukozo, saj so sevi spremenili fenotip porabe sladkorjev glede na kontrolni sev.

• Jezik: Slovenski jezik
• Ključne besede: kvasovke, proizvodnja piva, poraba sladkorjev, glukoza, maltoza, usmerjena evolucija, mutageneza
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Kraj izida: Ljubljana
• Založnik: [K. Ružič]
• Leto izida: 2022
• PID: 20.500.12556/RUL-139103
• UDK: 663.45:602.3:582.282.23:575.224.4
• COBISS.SI-ID: 119742723
• Datum objave v RUL: 31.08.2022

RUŽIČ, Katja, 2022, Sprememba fenotipa asimilacije sladkorjev v lokalnih izolatih kvasovk z usmerjeno evolucijo [na spletu]. Magistrsko delo. Ljubljana : K. Ružič. [Dostopano 8 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=139103

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Change of the sugar assimilation phenotype in local yeast isolates with directed evolution

Izvleček

One of the main problems when using local yeast isolates in beer production is their poor utilisation of alternative sugars, which directly affects the efficiency and speed of fermentation. Maltose is usually not consumed efficiently under catabolic repression in the presence of glucose, therefore it often remains unused at the end of the fermentation. The purpose of this master's thesis was to generate mutants resistant to 2-deoxyglucose (2-DOG) with application of directed evolution. 2-DOG is an antimetabolite of glucose that deregulates glycolysis and can indirectly forces yeast cell to activate the utilisation of alternative sugars. The main goal of this work was to abolish catabolic repression with glucose in selected 2-DOG resistant mutants and to improve maltose utilisation in two isolates Saccharomyces cerevisiae and S. paradoxus. In the first part of the thesis, the minimum inhibitory concentration of 2-DOG was determined on a defined medium with maltose. 2-DOG dependant survival curves were determined for both strains after the mutagen exposure. By applying directed evolution with mutagenesis, mutants resistant to 2-DOG and colonies that showed faster growth at higher concentrations of 2-DOG compared to the parent strain were isolated. The consumption of glucose, maltose, and ethanol during the fermentation was evaluated by applying HPLC method. We were able to abolish catabolic repression with glucose in selected mutants of S. cerevisiae, considering that the selected mutant strains utilised maltose and glucose simultaneously, in contrast to the parent strain.

• Ključne besede: yeasts, beer production, utilisation of sugars, glucose, maltose, directed evolution, mutagenesis