Using CRISPR/Cas9 to regulate enzyme expression levels in the Weimberg pathway for optimal xylose consumption in Saccharomyces cerevisiae

Abstract: Saccharomyces cerevisiae is a commonly used industrial organism which cannot consume xylose naturally. This is a problem since xylose is a major component in renewable resources, for instance lignocellulose. The Weimberg pathway is a pathway that converts xylose into α-ketoglutarate. It has been successfully integrated into S. cerevisiae, enabling it to utilize xylose. The successful strain had the genes xylB, xylD, xylX (from Caulobacter crescentus) and ksaD (from Corynebacterium glutamicum) integrated with one copy of xylB and four copies of each of the others. In addition, the strain had the iron regulon repressor gene, FRA2, deleted to enhance the activity of the XylD enzyme. In the present master thesis project, the aim was to find the optimal number of copies of the genes xylB, xylD, xylX and ksaD as well as what enzyme activities that are needed for the pathway to be functional. This was done by creating a pallet of strains that contained different copy numbers of the genes, using CRISPR/Cas9. Enzyme activity was measured before further analysis of biomass formation and metabolite production. It was found that TMB CB 016 (xylB, xylD, 4x(xylX, ksaD)) had an activity in the coupled assay, measuring the activity of XylD, XylX and KsaD together. However, due to the problems with the assay the results are unreliable and need to be further investigated. The constructed strains did not grow as well as the original strain with the functional Weimberg pathway, nor were any of them able to consume xylose.