Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae

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.

The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts

“…pastoris or S . cerevisiae ( Hamilton et al, 2006 ; Li et al, 2006 ; Jacobs et al, 2009 ; Parsaie Nasab et al, 2013 ; Piirainen et al, 2021 ). Importantly, these glycoproteins do not depend on glycans for proper folding and can be manufactured either as aglycones, in glycosylation incompetent host cells such as Escherichia coli or in vitro systems ( Higgins, 2010 ; Yin et al, 2012 ).…”

“…The Man 3 GlcNAc 2 glycan contains a terminal α-1,3 linked and α-1,6 linked mannose residue, respectively, that serve as acceptor for GnTI and GnTII ( Figure 2 , route D). This strategy has been utilized in H. polymorpha and S. cerevisiae ( Song et al, 2010 ; Parsaie Nasab et al, 2013 ; Piirainen et al, 2021 ).…”

“…In both organisms this was achieved by improving the flipping of the LLO into the ER lumen and in S. cerevisiae by additionally overexpressing a single-subunit OST ( Song et al, 2010 ; Parsaie Nasab et al, 2013 ). Furthermore, a number of mannosyltransferases compete with GnTI and GnTII for the Man 3 GlcNAc 2 glycan in the Golgi apparatus leading to glycan structures with additional mannose residues that precluded the complete processing into complex type N-glycans and reduce glycan homogeneity ( Song et al, 2010 ; Parsaie Nasab et al, 2013 ; Piirainen et al, 2016 , 2021 ).…”

The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells

“…Unfortunately, yeasts as a rule bind heterogeneous high-mannose glycan side chains on the recombinant glycoprotein, which may cause immunogenic reactions in humans, e.g., the typical α-1,3-linked mannose modifications of S. cerevisiae. For this reason, glycosylation pathways have been engineered in different yeast expression systems to yield human-like glycosylation patterns and thus avert the side effects of unwanted post-translational modifications in therapeutic proteins [36][37][38]. Although S. cerevisiae typically produces hyperglycosylated recombinant proteins, it is currently the most widely used host to produce yeasts-derived therapeutics [36,38].…”

Humanization of Yeasts for Glycan-Type End-Products

“…Och1(α-1,6-mannosyltransferase) and MNN1 (α-1,3-mannosyltransferase) produce mostly high-mannose glycans of the Man8GlcNAc2 ( Figure 1A ). Inactivation of the OCH1 gene is often an effective approach to eliminate hypermannosylation in H. polymorpha (Fukunaga et al, 2021b ; Gallo et al, 2022 ), K. lactis (Park et al, 2011 ), P. pastoris (Ma et al, 2019 ), S. cerevisiae (Piirainen et al, 2022 ), and Y. lipolytica (Karbalaei et al, 2020 ), which results in the expressed proteins with short-chain glycan from Man 3 GlcNAc 2 to Man 14 GlcNAc 2 ( Table 1 ). Especially in H. polymorpha , the deletion of OCH1 and alg3, and/or alg3 and alg11 can produce the protein with Man 3 GlcNAc 2 (Gündüz Ergün et al, 2019 ; Fukunaga et al, 2021b ).…”