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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. 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.

Aureobasidium pullulans je filamentozna gliva s kvasno obliko. Zaradi sposobnosti preživetja v ekstremnih pogojih je zanimiva za proučevanje in uporabo v industriji. Cilj magistrskega dela je bil pripraviti triptofanski avksotrofni sev Aureobasidium pullulans s sistemom CRISPR-Cas9, razvitim za uporabo v Aspergillus nidulans in nekaterih drugih filamentoznih glivah. Prekiniti smo želeli gen ApTRP1 z zapisom za encim fosforibozilantranil izomerazo, ki je tretji encim v sintezni poti triptofana. S prekinitvijo sinteze te esencialne aminokisline smo onemogočili rast in preživetje celice v odsotnosti triptofana. S sistemom CRISPR-Cas9 smo inducirali dvojni prelom DNA v predelu promotorja pred genom ApTRP1. V postopku popravljanja, usmerjenega s homologno rekombinacijo, je celica v genom vnesla zaporedje z zapisom za odpornost proti bleomicinu. Izbitje gena ApTRP1 oz. vgradnjo bleomicinske kasete smo zasledovali s protiselekcijo s 5-fluoroantranilno kislino, reakcijo PCR in rastjo na ploščah s oz. brez triptofana. Prekinjen gen ApTRP1 smo potrdili le z rastjo mutant na selekcijskem gojišču s 5-fluoroantranilno kislino. S primerjavo rasti na gojišču s triptofanom in brez njega ter reakcijo PCR izbitja ApTRP1 nismo uspeli potrditi. Aureobasidium pullulans is a yeast-like fungus. It thrives under extreme conditions and produces biotechnologically interesting products. The aim of the master's thesis was to prepare a tryptophan auxotrophic strain of Aureobasidium pullulans with the CRISPR-Cas9 system developed for genetic engineering of filamentous fungi. Our assignment was to knock-out the ApTRP1 gene. Its product is the third enzyme in the synthetic pathway of tryptophan. By interrupting the synthesis of this essential amino acid, the growth of the cell is inhibited in the absence of tryptophan. The CRISPR-Cas9 system induced a double-strand DNA break in the promoter region of the ApTRP1 gene. A double-strand break was repaired by homology-directed repair where a bleomycin cassette was integrated into A. pullulans genome. We used three different approaches to determine the knock-out of ApTRP1 gene: counterselection with 5-fluoroanranilic acid, PCR and the growth on plates with or without tryptophan. The gene knock-out was confirmed only with the growth on the plates with 5-fluoroanranilic acid. We did not confirm the mutants with the other two approaches.

Iz genske knjižnice poliekstremotolerantne črne kvasovke A. pullulans smo izbrali 4 različne gene – ACU1, ACU2, HAK in NHA, ki smo jih klonirali v celice S. cerevisiae s homologno rekombinacijo. Kot vektor smo uporabili plazmid pBEVY-U, ki ima kot selekcijski marker gen URA3. Plazmid smo imeli pomnožen v dveh delih, ki sta se prekrivala v selekcijskem markerju. Ob uspešni transformaciji so se fragmenti znotraj celice sestavili in selekcijski marker se je lahko izražal. Uspešne transformante smo nadaljnje testirali na gojiščih z različnimi stresnimi dejavniki. Rezultati so pokazali, da ATPaza P-tipa Acu1 omogoči rast pri visoki koncentraciji K+, črpalka Acu2 pa omogoči transformiranim celicam rast na gojiščih brez K+ ionov. Antiporterji Nha so transformiranim celicam omogočili rast na gojišču, ki je vsebovalo NaCl v koncentraciji 2 mol/L in transporterji Hak so izboljšali rast transformant na gojiščih s povišanimi koncentracijami glicerola (25 %) in LiCl (590 mM). Izvedli smo tudi in silico analize proteinskih zaporedij in rezultate primerjali z rezultati podobnih proteinov, ki smo jih dobili v bazah podatkov preko spleta. We selected four different genes from the gene library of plyextremotolerant black yeast A. pullulans and cloned them into cells of S. cerevisiae by homologous recombination. As a vector, we used plasmid pBEVY-U which has gene URA3 selection marker. The plasmid was multiplied in two parts with PCR so that they overlapped in the selection marker gene. In the case of successful transformation, fragments assembled within a cell and produced functional selection marker which could be expressed. After the transformation process we tested successfully transformed cells with few different stress factors – KCl, NaCl, LiCl, sorbitol and glycerol. The results showed that the Acu1 P-type ATPase improved growth of transformed cells in high K+ medium, whereas Acu2 ATPase allowed transformed cells to grow on the agar media with drastically reduced K+ content. Nha antiporters successfully improved yeast tolerance to NaCl, which was able to grow on medium containing NaCl at concentrations of 2 mol/L. Hak transporter have improved growth of transformed cells on medium containing up to 25 % glycerol and at high (590 mM) concentrations of LiCl. We have also performed some in silico analyses of protein sequences to gather more data about the chosen proteins.