N-Glycosylation Influences the Heterologous Expression of an Unspecific Peroxygenase From Marasmius rotula in Saccharomyces cerevisiae.

N-Linked glycosylation can have a significant impact on the yield of heterologously expressed proteins secreted by Saccharomyces cerevisiae. The yeast is a widely used host for the expression of unspecific peroxygenases (UPOs), a subclass of peroxide-dependent oxidoreductases with high potential for industrial biocatalytic applications. However, the effects of N-glycosylation on the expression of recombinant UPOs have not yet been investigated. Here, we studied respective protein modification on the expression of a UPO from Marasmius rotula, belonging to the protein subfamily of short peroxygenases, in S. cerevisiae. Two of the three N-glycosylation sites that are actually occupied in rMroUPO were eliminated by substituting asparagine at positions N43 and N151 with serine. The single substitutions led to reduced amounts of secreted rMroUPO, with N43S having the highest impact (almost four times lower protein amount compared to the native enzyme) and N151S having a moderate effect. In the next step, the glycosylation probability at position 43 was enhanced by replacing the serine in the corresponding sequon with threonine, which increased the expression of the rMroUPO variant. The concentration of active UPO in the concentrated culture supernatant of the glycosylation-optimised variant S45T was twice as high as that of the native rMroUPO. The results suggest that N-linked glycosylation is an important factor for successful heterologous UPO expression in S. cerevisiae.