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Author
Teixidó Barahona, Mònica
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Abstract
In recent years, glycolipids (GL) have attracted considerable attention as an immunotherapy strategy to activate the immune system. Α-Galactosylceramide has been shown to be able to bind to CD1d proteins to activate iNKT cells and rapidly secrete Th1 and Th2 cytokines. Therefore, these GLs are of great interest, although their synthesis is limited. Regarding the structure of this molecule, it consists of galactose linked by an α-glycosidic bond to a phytoceramide residue.
Yeasts have been used widely and successfully as host organisms for the production of useful substances in the fields of cosmetics, health foods, and medicine over the years. Saccharomyces cerevisiae is the most important genus of yeast from fundamental and applied perspectives and has been studied extensively. It also has the advantage that the two substrates necessary for the formation of α-galactosylceramide, UDP-galactose, and phytoceramide, are available and generated naturally by the yeast itself. All that is needed is to design a yeast that will accumulate large amounts of phytoceramide.
The main objective is to convert the yeast S.cerevisiae into a useful platform for the production of this GL and to be able to produce large concentrations. Therefore, complex lipid metabolism in S. cerevisiae was studied to reengineer ceramide biosynthetic pathways through the expression of key enzymes that redirect synthesis to the GL of interest. With this, the overexpression of the Sur2 and Isc1 genes was carried out and a knockout of the Scs7 gene was made.
Currently, it has been possible to obtain vectors for the overexpression of the two selected genes (Sur2 and Isc1 genes) and it was found that the two plasmids were functional in the yeast strain YM4271 by replacing the two genes with the mCherry protein. After this, a method for gene expression was established and 144 ± 36 overexpression was achieved with yeast strain YM4271 transformed with plasmid pTDH3_Sur2. The glycolipids were then extracted from each transformed strain with the mild-mild hydrolysis method and evaluated. On the other hand, for the KO of the Scs7 gene, all the necessary vectors have been designed (vector CAS9, plasmid donor DNA, and plasmid gRNA) and the yeast strain YM4271 has already been transformed with them.
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