Aureobasidium pullulans is a yeast-like fungus. It thrives under extreme conditions and produces biotechnologically interesting products. The aim of the master's thesis was to prepare a tryptophan auxotrophic strain of Aureobasidium pullulans with the CRISPR-Cas9 system developed for genetic engineering of filamentous fungi. Our assignment was to knock-out the ApTRP1 gene. Its product is the third enzyme in the synthetic pathway of tryptophan. By interrupting the synthesis of this essential amino acid, the growth of the cell is inhibited in the absence of tryptophan. The CRISPR-Cas9 system induced a double-strand DNA break in the promoter region of the ApTRP1 gene. A double-strand break was repaired by homology-directed repair where a bleomycin cassette was integrated into A. pullulans genome. We used three different approaches to determine the knock-out of ApTRP1 gene: counterselection with 5-fluoroanranilic acid, PCR and the growth on plates with or without tryptophan. The gene knock-out was confirmed only with the growth on the plates with 5-fluoroanranilic acid. We did not confirm the mutants with the other two approaches.
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