Molekularno-genetička karakterizacija konstruiranih sojeva kvasaca Scheffersomyces stipitis i Kluyveromyces marxianus te optimiranje metode transformacije Scheffersomyces stipitis

master's thesis

• Title (english): Molecular-genetic characterization of engeneered Scheffersomyces stipitis and Kluyveromyces marxianus a yeast strains and optimization of the Scheffersomyces stipites transformation method
• Author: Maja Horvat
• Mentor: Marina Svetec Miklenić (mentor)
• Committee member: Igor Slivac (predsjednik povjerenstva)
• Committee member: Ivan Krešimir Svetec (član povjerenstva)
• Committee member: Renata Teparić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2022-09-23, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Nekonvencionalni kvasci Kluyveromyces marxianus i Scheffersomyces stipitis imaju značajan potencijal kao proizvodni organizmi u različitim bioprocesima. S. stipitis jedan je od kvasaca s najvišom sposobnošću fermentacije ksiloze u etanol, dok K. marxianus osim što je termofilan, brzo raste i može koristiti širok spektar različitih supstrata. Međutim u oba kvasca uvođenje ciljnih genetičkih modifikacija otežano je zbog česte ilegitimne (nehomologne) rekombinacije. Prvi cilj ovog rada bila je molekularno-genetička karakterizacija prethodno konstruiranih transformanata K. marxianus i S. stipites, koji proizvode L-laktat i 2,3-butanediol, metodom hibridizacije po Sothern-u. U kvascu S. stipitis istražena je mogućnost transformacije metodom elektroporacije i metodom protoplastiranja te utjecaj koncentracije ribonukleinskog kompleksa pri korištenju sustava CRISPR/Cas9. Dobiveni rezultati ukazuju na zaključak da prethodno razvijena metoda protoplastiranja nije pogodna za transformaciju kvasca S. stipitis te da koncentracija ribonukleinskog kompleksa značajno utječe na uspješnost transformacije elektroporacijom.

Abstract (english)

Non-conventional yeasts Kluyveromyces marxianus and Scheffersomyces stipitis have significant potential as production organisms in various bioprocesses. S. stipitis is one of the yeasts with the highest ability to ferment xylose into ethanol, while K. marxianus, in addition to being thermophilic, grows quickly and can use a wide range of different substrates. However, in both yeasts, the introduction of target genetic modifications is difficult due to frequent illegitimate (non-homologous) recombination. The first goal of this work was the molecular-genetic characterization of previously constructed transformants of K. marxianus and S. stipites, which produce L-lactate and 2,3-butanediol, using the Southern blotting hybridization method. In the yeast S. stipitis, the possibility of transformation by the electroporation method and the protoplasting method and the influence of the ribonucleic complex concentration when using the CRISPR/Cas9 system were investigated. The obtained results indicate the conclusion that the previously developed protoplasting method is not suitable for the transformation of the yeast S. stipitis and that the concentration of the ribonucleic complex significantly affects the success of the transformation by electroporation.

• Keywords: Scheffersomyces stipitis; Kluyveromyces lactis; nekonvencionalni kvasci; genetička transformacija
• Keywords (english): Scheffersomyces stipitis; Kluyveromyces lactis; non-conventional yeasts; genetic transformation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:443601
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-03-01 11:00:19