Mutagenesis and directed evolution are non-recombinant techniques used to improve industrial strains of microorganisms in the food industry. In polyploid yeast strains, allelic variability occurs, which causes concerns that positive mutations will be lost through repair by homologous recombination. This can be avoided by sporulating the improved mutants, resulting in a haploid spore state that loses allelic variability after auto-diploidization. In doing so, we must be careful to maintain the desired phenotype in the sporulated mutants. The aim of the master 's thesis was to determine whether monosporic yeasts have preserved the altered maltose uptake phenotype. Sporulation was achieved by restricting the nutrients in the yeast cultivation medium by removing the nitrogen source. The yeasts were first, grown on nutrient-rich medium and then they were transferred onto acetate medium lacking nitrogen source, where they formed ascospores. To degradate their cell wall, we prepared tetrad juice with lyticase and separated the released ascospores with a micromanipulator. We determined the ability of monosporic cultures to ferment maltose in 2-mL microtubes and selected cultures also in 50-mL tubes. The kinetics of sugar consumption and ethanol formation during fermentation were determined by HPLC analysis. We found that some monosporic cultures retained the altered sugar uptake phenotype also in large scale. In the last part of the work, we determined the mating type based on the length of the PCR amplified MAT locus of monosporic cultures, which showed that most of the monosporic yeast cultures were auto-diploidized.
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