University of Natural Resources and Life Sciences, Vienna (BOKU) - Research portal
Gewählte Master / Diploma Thesis:
Julia Prinz
(2020):
Genetic tools development in the thermophilic fungus Thermoascus aurantiacus for cost-efficient conversion of plant biomass.
Master / Diploma Thesis - Institut für Pflanzenbiotechnologie und Zellbiologie (IPBT),
BOKU-Universität für Bodenkultur,
pp 108.
UB BOKU
obvsg
- Abstract:
- Fungal enzymes such as cellulases and xylanases can deconstruct non-edible plant biomass (lignocellulose) into simple sugars which can then be converted to biofuels, bioplastics or other bio-products. Thermoascus aurantiacus is an attractive host because this thermophilic fungus produces enzymes with higher activity and thermotolerance than many industrially used enzymes. The use of T. aurantiacus enzymes would allow more cost-efficient deconstruction of plant biomass. Since there is currently a lack of effective, versatile genome-editing methods, the aim of this Master thesis was to develop new genetic tools to aid strain engineering for improved enzyme production. Therefore, the CRISPR/Cas9 technology was established for the first time in T. aurantiacus and a sexual crossing protocol was developed due to the self-fertilizing ability of this fungus. These approaches will increase the fundamental understanding of how enzyme secretion is regulated in this fungus and enable the design of improved strain and bioprocess engineering strategies. We demonstrated the functionality of the CRISPR/Cas9 system in T. aurantiacus using pyrG as target gene and in model organism Aspergillus niger using albA as target gene for editing through Agrobacterium tumefaciens-mediated transformation. The generated CRISPR/Cas9 constructs introduced site-specific mutations in the target genes. Sexual crosses were set up using a hygromycin resistant and an uracil auxotrophic 5-fluoroorotic acid resistant T. aurantiacus strain resulting in a mutant harboring both resistances. In conclusion, two genome editing methods were successfully established for T. aurantiacus which can be used individually or in combination and have the potential to accelerate strain engineering of the thermophilic fungus to produce industrial enzymes like cellulases and possibly biofuels in the future.
- Beurteilende*r: Mach Lukas