Heterologno izražanje genov za zunajcelične encime glive Aureobasidium pullulans v kvasovki Saccharomyces cerevisiae

Kustec, Anita (author), Gunde Cimerman, Nina (mentor)

Aureobasidum pullulans je vrsta črne kvasovke, ki je znana predvsem po svoji biotehnološki vlogi kot proizvajalec polisaharida pululan in cele vrste zunajceličnih encimov. Izraba le-teh je zaenkrat omejena, vendar nedavna določitev genomskega zaporedja vrste A. pullulans predstavlja izjemno priložnost za identifikacijo in preučitev encimov te glive. Za A. pullulans je značilna tudi toleranca na različne stresne pogoje kot so nizka vodna aktivnost, visoke koncentracije soli, nizek pH. Pivska ali pekovska kvasovka Saccharomyces cerevisiae je uveljavljen laboratorijski modelni sistem. Ker vrsta S. cerevisiae ni patogena, lahko z njo brez težav rokujemo v mikrobiološkem laboratoriju ob običajnih varnostnih ukrepih. Kvasovka je pomembna v živilski industriji pri vzhajanju testa in proizvodnji alkoholnih pijač, pa tudi v energetiki za proizvodnjo bioetanola za gorivo, zaradi česar je spreminjanje njenih industrijsko relevantnih lastnosti zelo zaželeno. Namen magistrskega dela je bil izbrati gene za zunajcelične encime, ki imajo potencial za uporabo v industriji. Namen dela je bil tudi izražanje teh genov v laboratorijskem sevu pivske kvasovke in njihova osnovna karakterizacija. Pri raziskovalnem delu smo najprej identificirali gene za encime A. pullulans. Sledilo je pomnoževanje teh genov z verižno reakcijo s polimerazo in ločevanje pomnožkov z agarozno gelsko elektroforezo. Naslednja stopnja je bilo čiščenje pomnožkov in vstavljanje v plazmid pBEVY-U. Sledila je transformacija S. cerevisiae. Dobljene transformante smo preverili z verižno reakcijo s polimerazo in z določanjem nukleotidnega zaporedja vstavljenih genov. V naslednji stopnji smo preverjali funkcijo vstavljenih genov na gojiščih za zaznavo ustreznih encimskih aktivnosti. Nazadnje pa smo preverili delovanje izbranih encimov med rastjo gensko spremenjene kvasovke S. cerevisiae v stresnih pogojih. A. pullulans je namreč znana kot ekstremnotoleranten organizem, ker raste tudi pri povišani koncentraciji soli in pri različnih temperaturah. Zato smo delovanje zunajceličnih encimov, za katere smo zapise vnesli v S. cerevisiae, preverjali pri povišani slanosti in pri različnih temperaturah. Gene za zunajcelične encime A. pullulans smo uspeli izraziti v S. cerevisiae. Nenazadnje pa smo uspeli dokazati, da encimi iz A. pullulans delujejo tudi v stresnih pogojih ob dodatku natrijevega klorida v gojišče.

• Language: Slovenian
• Keywords: Zunajcelični encim, gen, Aureobasidium pullulans, Saccharomyces cerevisiae
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-113456
• COBISS.SI-ID: 1538480067
• Publication date in RUL: 08.01.2020

KUSTEC, Anita, 2019, Heterologno izražanje genov za zunajcelične encime glive Aureobasidium pullulans v kvasovki Saccharomyces cerevisiae [online]. Master’s thesis. [Accessed 28 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=113456

Secondary language

• Language: English
• Title: Heterologous gene expression for Aureobasidium pullulans extracellular enzymes in yeast Saccharomyces cerevisiae

Abstract

Aureobasidum pullulans is a black yeast, particularly known for its biotechnological role in the production of the polysaccharide pullulan and of many extracellular enzymes. The use of these enzymes is currently limited, but recent sequencing of the genome of A. pullulans represents a good opportunity for identification and examination of its enzymes. A. pullulans has high tolerance for stress conditions like low water activity, high salt concentrations and low pH. The yeast Saccharomyces cerevisiae is a well-established laboratory model system. Because S. cerevisiae is not pathogenic, we can handle with it in a microbiological laboratory without special precautions. Baker’s yeast is important in the food industry for rising the dough and in the alcohol drink industry. It is also important in energetics, where it is particularly used for production of bioethanol. For all these reasons S. cerevisiae is extensively studied and modifications, which would improve its biotechnologically important traits, are constantly being attempted. The purpose of this master thesis was to select genes for extracellular enzymes with potential industrial use. Furthermore, the purpose was to express these genes in the laboratory strain of S. cerevisiae and their basic characterization. In our research, we first identified genes for enzymes and amplified genes. Amplified genes were separated with agarose gel electrophoresis. The next step was purification of the amplifiers and insertion into the plasmid pBEVY-U. The transformation of S. cerevisiae followed. The transformants were verified by polymerase chain reaction and by determining the nucleotide sequence of the inserted genes. In the next step, the function of the inserted genes on the media was checked to detect the corresponding enzyme activities. Finally, we tested the activity of selected enzymes during the growth of genetically modified yeast under stress conditions. A. pullulans is known as an extremotolerant organism, it also grows at elevated salt concentrations and at different temperatures. Therefore, the activity of extracellular enzymes was checked at elevated salinity and at different temperatures. We were able to express the genes for the extracellular enzymes of A. pullulans in S. cerevisiae. Last but not least, we were able to demonstrate that extracellular enzymes from A. pullulans also function under stress conditions with the addition of sodium chloride in culture medium.

• Keywords: Extracellular enzyme, gene, Aureobasidium pullulans, Saccharomyces cerevisae