20.500.12556/RUL-184369 Uporaba toksičnega proteina kot senzorja uspešnosti izločanja proteinov v kvasovki Saccharomyces cerevisiae Use of a toxic protein as a sensor for protein secretion efficiency in yeast Saccharomyces cerevisiae V industrijski biotehnologiji je pomemben izziv razvoj sevov mikroorganizmov z izboljšano sposobnostjo izločanja rekombinantnih proteinov. Kvasovka Saccharomyces cerevisiae je pogosto uporabljen organizem v biotehnologiji zaradi svoje genetske organiziranosti in enostavnega gojenja, vendar je njena učinkovitost izločanja proteinov v primerjavi z drugimi sistemi omejena. V sklopu magistrske naloge smo želeli razviti metodo, ki bi omogočila razvoj seva S. cerevisiae z izboljšanim izločanjem proteinov. Za razvoj takšne metode smo uporabili endogene toksične proteine in signalni peptid feromona α-faktorja. Najprej smo izbrali toksične proteine, katerih prekomerno izražanje vodi v zmanjšano viabilnost celic. Osredotočili smo se na protein Arf1, ki sodeluje pri vezikularnem transportu, in Kar2, ki je pomemben šaperon v endoplazemskem retikulumu. Z uporabo različic promotorja GAL1 smo uravnavali njuno izražanje ter preučili vpliv signalnega peptida α-faktorja na njuno sekrecijo. Pričakovali smo, da bo usmeritev proteina v sekretorno pot zmanjšala njegovo toksičnost in omogočila selekcijo tistih sevov, ki protein najbolj učinkovito izločajo. V eksperimentalnem delu smo uporabili tehniko CRISPR-Cas9 za vstavitev skrajšanih različic promotorja GAL1 in zapisa za signalni peptid α-faktorja v genom sevov S. cerevisiae. Nastale mutante smo analizirali s fenotipskimi testi, določanjem hitrosti rasti ter analizo proteinov z SDS-PAGE in prenosom western. Ugotovili smo, da protein Kar2 v uporabljenem sistemu ne deluje toksično. Nasprotno pa protein Arf1 kaže toksičen učinek, če je prekomerno izražen. Fuzija Arf s signalnim zaporedjem α-faktorja, na celico ni imela toksičnega učinka, vendar pa smo pokazali tudi, da sekrecija proteina ni bila uspešna. Rezultati kažejo, da je izbira toksičnega proteina ključna za delovanje sekrecijskega sistema. Čeprav je Arf1 sprva deloval kot potencialen kandidat, se je izkazalo, da dodatek signalnega zaporedja ne zadostuje za njegovo učinkovito izločanje. Nadaljnje raziskave bi lahko vključevale prilagajanje signalnih zaporedij in optimizacijo sekrecijske poti za specifične proteine, kar bi še dodatno izboljšalo učinkovitost biotehnoloških procesov. In industrial biotechnology, a key challenge is the development of microbial strains with enhanced secretion capabilities for recombinant proteins. The yeast Saccharomyces cerevisiae is a widely used organism in biotechnology due to its genetic accessibility and ease of cultivation. However, its secretion efficiency is limited compared to other systems. In this master's thesis, we aimed to develop a method for selecting yeast strains with improved protein secretion. To achieve this, we utilized endogenous toxic proteins and the signal peptide of the alpha-factor pheromone. First, we selected toxic proteins whose overexpression leads to reduced cell viability. We focused on the Arf1 protein, which is involved in vesicular transport, and Kar2, a key chaperone in the endoplasmic reticulum. By using different versions of the GAL1 promoter, we regulated their expression and studied the effect of the alpha-factor signal peptide on their secretion. We hypothesized that directing the toxic protein into the secretory pathway would reduce its toxicity and enable the selection of strains that most efficiently secrete the protein. For the experimental part, we used the CRISPR-Cas9 technique to insert shortened versions of the GAL1 promoter and the alpha-factor signal peptide sequence into the genome of S. cerevisiae strains. The resulting mutants were analysed using phenotypic assays, growth measurements, and SDS-PAGE and Western blot protein analyses. We found that the protein Kar2 is not toxic in the system used. In contrast, the protein Arf1 exhibits a toxic effect when overexpressed. Fusion of Arf1 with the α-factor signal sequence did not have a toxic effect on the cell, however, we also demonstrated that secretion of the protein was not successful. These results indicate that the choice of a toxic protein is critical for the functionality of the secretion system. Although Arf1 initially appeared to be a promising candidate, it was shown that the addition of a signal sequence alone is not sufficient for its efficient secretion. Further research could include fine-tuning signal sequences and optimizing the secretion pathway for specific proteins, further improving the efficiency of biotechnological processes. Saccharomyces cerevisiae toksični proteini sekrecija signalni peptid α-faktorja promotor GAL1 CRISPR-Cas9 Saccharomyces cerevisiae toxic proteins secretion alpha-factor signal peptide GAL1 promoter CRISPR-Cas9 true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-06 09:36:49 2026-07-06 09:36:59 2026-07-31 12:31:11 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 23091 286586371 Gabric_Masa_-_Uporaba_toksicnega_proteina_kot_senzorja_uspesnosti_izlocanja_proteinov_v_kvasovki.pdf Gabric_Masa_-_Uporaba_toksicnega_proteina_kot_senzorja_uspesnosti_izlocanja_proteinov_v_kvasovki.pdf 1 6CAB95E2FF874AFDF0E6C433820DBDB7 1430092ef175c97d92977a8bbc95761f66ad56fd112eb2f23affc824c549fa1f 5b1eab24-790d-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237052 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0