CRISPR/Cas9 för introduktion av metaboliska vägar som möjliggör konsumtion av ättiksyra och xylos i Saccharomyces cerevisiae
Examensarbete för kandidatexamen
Hammer úr Skúoy, Pauline
Abstract CRISPR/Cas9 for introduction of metabolic pathways to enable the consumption of acetic acid and xylose in Saccharomyces cerevisiae An alternative to fossil fuel is the usage of Saccharomyces cerevisiae in the process of producing bioethanol from lignocellulosic biomass. However, S. cerevisiae can only utilise around 70% of the sugars from lignocellulose to produce ethanol, rendering the process inefficient. In order to increase the use of the sugars present in lignocellulosic biomass, this study aimed to introduce metabolic pathways for both acetic acid and xylose consumption in S. cerevisiae and analyse the effects of the modifications. For the acetic acid consumption the adhE-gene was introduced whereas for the xylose consumption three genes were introduced encoding xylose reductase (XR), xylitole dehydrogenase (XDH), and xylulokinase (XK). The CRISPR/Cas9 genome editing system was transformed into the cells to introduce the two metabolic pathways seperately in 12 strains of S. cerevisiae. The 12 strains included both laboratory strains as well as industrial and wild type strains. To analyse the results the three methods colony PCR, growth on solid selective media, and growth on liquid selective media were used. All studied strains had successful transformations for both pathways. On the other hand, the number of colonies varied between the different strains. For both pathways, the laboratory strains generally had more transformed colonies than the industrial and wild type strains. The lack of an integrated selection marker when using CRISPR/Cas9 as the genome editing tool made the selection of successfully engineered strains difficult. However, the results from growth in liquid media and colony PCR indicated that the integration of the metabolic pathway was successful for some of the strains. These results show that CRISPR/Cas9 is an useful method for integrating new metabolic pathways in S. cerevisiae and with further investigations the strains could provide important information in the development of bioethanol production.
Livsvetenskaper , Mikrobiologi , Kemiska processer , Mikrobiologi inom det medicinska området , Life Science , Microbiology , Chemical Process Engineering , Microbiology in the medical area