Abstract
N-glycosylation is an important posttranslational modification affecting the properties and quality of therapeutic proteins. Glycoengineering in yeast aims to produce proteins carrying human-compatible glycosylation, enabling the production of therapeutic proteins in yeasts. In this work, we demonstrate further development and characterization of a glycoengineering strategy in a Saccharomyces cerevisiae Δalg3 Δalg11 strain where a truncated Man3GlcNAc2 glycan precursor is formed due to a disrupted lipid-linked oligosaccharide synthesis pathway. We produced galactosylated complex-type and hybrid-like N-glycans by expressing a human galactosyltransferase fusion protein both with and without a UDP-glucose 4-epimerase domain from Schizosaccharomyces pombe. Our results showed that the presence of the UDP-glucose 4-epimerase domain was beneficial for the production of digalactosylated complex-type glycans also when extracellular galactose was supplied, suggesting that the positive impact of the UDP-glucose 4-epimerase domain on the galactosylation process can be linked to other processes than its catalytic activity. Moreover, optimization of the expression of human GlcNAc transferases I and II and supplementation of glucosamine in the growth medium increased the formation of galactosylated complex-type glycans. Additionally, we provide further characterization of the interfering mannosylation taking place in the glycoengineered yeast strain.Key points• Glycoengineered Saccharomyces cerevisiae can form galactosylated N-glycans.• Genetic constructs impact the activities of the expressed glycosyltransferases.• Growth medium supplementation increases formation of target N-glycan structure.
Highlights
Therapeutic proteins are a fast-growing product segment in the pharmaceutical industry, consisting of products such as antibodies, hormones, and vaccines
We have previously reported that complex-type glycans bearing terminal GlcNAc residues can be generated in S. cerevisiae and that their amount can be increased by enhancing the transport of UDP-GlcNAc to the Golgi apparatus (Parsaie Nasab et al 2013; Piirainen et al 2016)
We have previously shown that complex-type GlcNAc2Man3GlcNAc2 (G0) glycans can be formed in a glycoengineered Δalg3 Δalg11 S. cerevisiae strain and that its amount can be increased by the expression of a UDPGlcNAc transporter from K. lactis (Yea4) (Parsaie Nasab et al 2013; Piirainen et al 2016)
Summary
Therapeutic proteins are a fast-growing product segment in the pharmaceutical industry, consisting of products such as antibodies, hormones, and vaccines. The increasing production of therapeutic proteins can provide opportunities to develop alternative production platforms for the predominantly mammalian cell-based production processes. Yeasts have many advantages over mammalian cells in biotechnological processes, including. A significant proportion of therapeutic proteins contain N-glycans. N-glycosylation is a heterogeneous and speciesspecific posttranslational modification, and the presence of N-glycans as well as their structures can have a significant impact on the properties of a protein. The glycan pattern in antibodies can affect their therapeutic efficacy (Kurogochi et al 2015; Reusch and Tejada 2015). The differences in the native N-glycosylation between yeast and mammalian cells currently prevent the use of yeast for the production of therapeutic glycoproteins, as nonhuman
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