In the master's thesis we investigated the possibility of improving the alkali tolerance of selected fungi using the adaptive laboratory evolution (ALE) method, with the aim of obtaining strains capable of survival and biomineralization of concrete. From the Ex collection (MRIC UL) we selected 35 strains with naturally expressed urease activity, ability to sporulate, and the potential to biomineralize calcium carbonate, of which the strain Aureobasidium sp. EXF-16720 was selected for further experiments. In 8 consecutive ALE cycles, the culture was exposed to a gradual increase in pH from 7 to 10,5. Phenotypic changes were monitored, and genome sequencing was performed for the first and last two cycles of evolution. The sequences were assembled into scaffolds using bwa software, variant calling was done using gatk4 and the genes affected by the mutations were annotated using Augustus. After ALE the strain Aureobasidium sp. EXF-16720 was able to grow at a pH of 10.5, which represents a significant improvement compared to the original strain. At the same time, it retained the ability to biomineralise. This was confirmed by cultivation on a biomineralization medium, which was followed by the isolation of crystals. Analysis by scanning electron microscopy, energy-dispersive spectroscopy, and Raman spectroscopy was performed on the acquired crystal samples. Preliminary characterization showed presence of calcite and calcium phosphates. Genomic analyses revealed mutations related to urea transport, that matched acquired phenotypic traits. We have thus confirmed the hypotheses on the effectiveness of ALE in improving alkali tolerance and biomineralization potential of selected fungi. The findings contribute to furthering the possibilities of using fungi as upstart building blocks of a new generation of bioconcrete and enable further research to improve the strains and their application potential.
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