Cell factory development of Aspergillus saccharolyticus for the production of bovine beta-lactoglobulin
Author
Szabados, Flóra Anna
Term
4. term
Education
Publication year
2026
Submitted on
2026-08-10
Abstract
This project investigates whether the filamentous fungus A. saccharolyticus can be used as a fungal cell factory to produce the protein β-lactoglobulin (βLG). The aim is to develop a more sustainable way to make protein products by combining fungal production with biorefinery approaches that use biological raw materials more efficiently. To enable genetic modification of the fungus, a workflow was developed and optimized for generating protoplasts (fungal cells without a cell wall) and introducing new DNA into them via PEG-mediated transformation. This workflow produced stable, reproducible, high-quality protoplasts and showed that foreign DNA can be successfully taken up by the cells. Diagnostic tests confirmed that the AMA1 plasmid was maintained episomally and stably, meaning it remained in the cells without integrating into the genome; this is the first reported demonstration of AMA1 functionality in A. saccharolyticus. In addition, a five-fragment βLG expression vector was successfully assembled using Gibson Assembly and verified by DNA sequencing. However, the attempt to generate a ∆ku70 deletion mutant did not succeed. Post-hoc in silico analysis revealed that the two ku70 spacer oligonucleotides had been cloned in the wrong orientation, which made the corresponding CRISPR-Cas9 guide RNAs non-functional.
Dette projekt undersøger, om den trådformede svamp A. saccharolyticus kan bruges som en såkaldt svampe-cellefabrik til at producere proteinet β-lactoglobulin (βLG). Målet er at skabe en mere bæredygtig måde at fremstille proteinprodukter på ved at koble svampeproduktion sammen med bioraffinering, hvor biologiske råvarer udnyttes mere fuldt. For at kunne ændre svampens arvemateriale blev der udviklet og optimeret en metode til at fremstille protoplaster (svampeceller uden cellevæg) og til at indføre nyt DNA i dem ved hjælp af PEG-medieret transformation. Denne metode gav stabile og reproducerbare protoplaster og viste, at fremmed DNA faktisk kunne optages af cellerne. Der blev udført diagnostiske analyser, som viste, at AMA1-plasmidet kunne opretholdes stabilt i cellerne uden at integreres i arvematerialet; dette er den første dokumenterede demonstration af AMA1-funktionalitet i A. saccharolyticus. Derudover blev et fem-fragment βLG-udtryksvektor konstrueret ved Gibson Assembly og bekræftet ved sekventering. Forsøget på at skabe en ∆ku70-sletningsmutant lykkedes dog ikke. Efterfølgende computerbaseret analyse viste, at de to ku70-spacer-oligonukleotider var klonet i omvendt orientering, hvilket gjorde de tilhørende guide-RNA’er til CRISPR-Cas9 ikke-funktionelle.
[This abstract has been rewritten with the help of AI based on the project's original abstract]
Keywords