Yeast Saccharomyces cerevisiae is a unicellular eukaryote widely used for recombinant protein production and as a model organism for investigation of various cellular processes including protein secretion and glycosylation in lower and higher eukaryotes. Protein glycosylation is an enzymatic oligosaccharide attachment to protein amino acids. Dolichol kinase (DK), encoded by the SEC59 gene in S. cerevisiae, is essential for core steps of protein glycosylation. DK resides in the membrane of endoplasmic reticulum where it phosphorylates dolichol. Synthesized dolichol phosphate is then used as a carrier on which the core oligosaccharide is assembled and transferred on protein in the process of its glycosylation. Such glycoproteins are then further modified in the Golgi apparatus and highly diverse glycoproteins, needed for various cellular functions are produced [1].
Although core steps of protein glycosylation in different yeast species are very similar, some significant functional differences also exist. It was shown that reduced DK activity of temperature-sensitive S. cerevisiae DK mutant sec59-1 results in decreased secretion and glycosylation of both invertase and carboxypeptidase Y (CPY), alterations of cell wall structure and accumulation of hypoglycosylated proteins in its endoplasmic reticulum [2, 3]. Kluyveromyces lactis DK mutant WSS also demonstrated protein glycosylation deficiencies but this had fewer effects on yeast cell wall integrity and even improved secretion of some recombinant proteins compared to wild type cells [4].
The aim of this research was to investigate the effect of different DK mutations on protein glycosylation process in yeast S. cerevisiae. For introduction of mutations into SEC59 gene we used components of bacterial adaptive immunity system CRISPR-Cas9 which became one of the most widely used genome editing tools and has been applied to modify yeast genomes as well. Employment of this system allowed to edit the genome of S. cerevisiae with the effectiveness close to 100 % and without the use of any selective markers [5]. We introduced mutations, encoding W332G, G407S, G420D, L421S and double G407S and L421S substitutions in DK amino acid sequence, into SEC59 gene of S. cerevisiae and investigated the CPY glycosylation ability as well as sensitivity to tunicamycin and fluorescent Calcofluor white dye (CFW) of all five constructed yeast strains. Our results showed that the degree of CPY glycosylation deficiency correlated with sensitivity of constructed mutants to tunicamycin and temperature and these changes were most pronounced in double DK mutant, similar to K. lactis WSS mutant with the corresponding mutations. Nonetheless, the decrease in DK activity in S. cerevisiae double mutants, in contrast to K. lactis mutants, had impact on yeast cell wall structure, as mutant cells became sensitive to CFW. It is possible, that the decrease in DK activity in S. cerevisiae, more strongly than in K. lactis affects the cell wall protein composition through changes of lipid metabolism and/or GPI glycosylation.