P80: Production of glycosylation-free protein from glycoengineered Pichia pastoris

We have expressed a non-glycosylated human recombinant protein from a genetically modified strain of Saccharomyces cerevisiae (Sc).  The undesired O-glycosylation of heterologous recombinant protein in this yeast was successfully resolved by disrupting the pmt1 gene.  When evaluating Pichia pastoris (Pp) as a potential alternate expression system for production of heterologous proteins, the same undesired O-linked glycosylation was observed at multiple residues on the molecule.  In Saccharomyces cerevisiae there are 7 PMT genes which control the first step in the glycosylation pathway.  Based on the recently completed Pichia genome database several PMT homologues were found (PMT1, 2, 3, 4 and 5).  Pp PMT1 shares 53% homology with the Sc PMT1 gene.  However, disrupting the Pp pmt1 gene in Pichia did not prevent the undesired glycosylation of human recombinant protein.  During the investigation of the Pp PMT gene family we observed that a single knockout of either pmt1 or pmt4 mutant resulted in dramatic cell death in zymolyase treatment, suggesting the equal importance of PMT1 and PMT4 in maintaining a cell wall integrity.  A pmt4 deficient mutant successfully resulted in the production of heterologous recombinant protein which did not have the undesired glycosylation.  This work demonstrates that glycoengineered Pichia can be used in producing non-glycosylated or special desired glycosylated recombinant proteins.