CRISPR/Cas9 system implementation in Torulaspora delbrueckii

Student thesis: Master thesis (including HD thesis)

  • Lev Koval
4. term, Sustainable Biotechnology, Master (Master Programme)
For the long time Saccharomyces cerevisiae has been used as the main microorganism which in industrial and food biotechnology. It was proven that it is an efficient ethanol producer and can tolerate high amounts of ethanol produced during fermentative processes. S. cerevisiae, as well as the other yeasts, also produces fusel alcohols such as: 2-phenylethanol, tryptophol and tyrosol. Importance of fusel alcohols is undeniable since they have a large application in different industries. Torulaspora delbrueckii is the perfect candidate for the
2-phenylethanol production since it can produce the same or even more amounts of 2-phenylethanol under certain conditions. Numerous genetic manipulations were applied in S. cerevisiae in order to increase 2-phenylethanol production. Mostly genes involved in Ehrlich pathway were either disrupted or overexpressed from which overexpression of ARO10 and disruption of PDC genes were highlighted. On the other hand, T. delbrueckii could serve as a potential ethanol producing organism if specific modifications such as RIM15 gene disruption are considered, where RIM15 is the gene responsible for ethanol production inhibition. However, unlike S. cerevisiae, T. delbrueckii lacks of sufficient genetic manipulation tools for efficient metabolic engineering. During this project it was aimed to construct a toolkit with the possibility of utilization of CRISPR/Cas9 system to obtain desired modifications of PDC gene disruption and ARO10 overexpression for better 2-phenylethanol production in one case and RIM15 disruption to increase ethanol production in another case. For this purpose, EasyClone - Markerfree toolkit constructed for S. cerevisiae was used as a model. It was also aimed to investigate the requirement of integration cassette homologous sites size for successful integration of the gene of interest into desired genomic location. For this purpose 3 integration cassettes containing 50, 200 and 500 base pairs of homology to URA3 gene in T. delbrueckii were constructed. It was shown that the highest chance of integrating the gene of interest requires 500 base pairs of homology to the desired genomic location. Moreover, it was shown that supposedly 70 base pair homology and/or low concentration of supplied integration cassette was not enough to introduce ARO10 and disruption cassette to desired location for PDC gene disruption. Further investigation is required in order to understand how and where RIM15 integration cassette containing 200 base pair homology was integrated into genome.
LanguageEnglish
Publication date10 Jun 2019
Number of pages55
ID: 305539842