Engineering of N-glycosylation in Saccharomyces cerevisiae

N-glycosylation is a post-translational modification with a significant impact on the properties of therapeutic proteins. Most therapeutic proteins are currently produced using mammalian cells due to the requirement of correct post-translational modifications, especially N-glycosylation. Yeast Saccharomyces cerevisiae is a commonly utilized organism with many advantages over mammalian cells. However, the N-glycan structures generated in yeasts are not suitable for therapeutic use, preventing the utilization of yeast for the production of therapeutic glycoproteins. Engineering of the N-glycosylation pathway in yeast could overcome this challenge. Although different yeast glycoengineering approaches have been developed, efficient human-compatible glycosylation has not yet been obtained in S. cerevisiae, and in-depth knowledge is lacking on the consequences of glycoengineering on the quality and quantity of the produced protein. This work aims to further develop a glycoengineering approach based on ALG3 and ALG11 deletions in S. cerevisiae and to gain better understanding of the connection between glycoengineering and intracellular protein quality control. First, the interfering mannosylation taking place in the glycoengineered Δalg3 Δalg11 S. cerevisiae strain could be partially eliminated by the deletion of MNN1 gene. In addition, optimization of the expression constructs of human GnTI and GnTII, coexpression of a UDP-GlcNAc transporter and supplementation of glucosamine into the growth medium improved the efficiency of complex-type glycan formation. The introduction of galactose residues to hybrid-like and complex-type glycans via the expression of a human GalTI fused to a galactose epimerase domain from Schizosaccharomyces pombe was additionally demonstrated. Second, the impact of glycoengineering on intracellular protein quality control was studied from the perspective of recombinant protein production. The glycoengineered Δalg3 Δalg11 strain generates a Man3GlcNAc2 glycan intermediate that lacks the glycan structures normally serving as recognition signals for the endoplasmic reticulum protein quality control system. The impact of glycoengineering on the endoplasmic reticulum-associated protein degradation (ERAD) pathway was investigated by monitoring the impact of ALG3, ALG11 and ERAD gene deletions on intracellular processing of an IgG model protein. In Δalg3 Δalg11 strain, intracellular IgG was targeted for ERAD independently of Yos9p and Htm1p. Upon expression of ALG3, ERAD targeting was dependent on Yos9p but Htm1p was not required. Our findings suggest that ERAD targeting of a protein can occur via other mechanisms than the established Htm1p and Yos9p-dependent route when the N-glycan biosynthesis pathway is altered. Although several challenges remain to be tackled, glycoengineered S. cerevisiae has potential to become an alternative production platform for therapeutic proteins and enable production of glycoproteins with human-compatible N-glycosylation pattern.