Identification and functional characterization of the HpALG11 and the HpRFT1 genes involved in N-linked glycosylation in the methylotrophic yeast Hansenula polymorpha

Abstract

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). In this study, we have identified two genes, HpALG11and HpRFT1, in the metylotrophic yeast Hansenula polymorpha. Detailed analysis of the glycan structures of the N-linked glycans of secreted recombinant glucose oxidase in mutant strains Hpalg3Δ, Hpalg11Δ, and Hpalg3Δalg11Δ with the assistance of over-expression of RFT1 was performed by linkage-specific mannosidase digestion. The results suggest that HpALG11 and HpRFT1 were responsible for catalyzing the sequential transfer of terminal α-1,2-Man residues to form the Man(5)GlcNAc(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side and encoding an evolutionarily conserved protein required for the translocation of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane, respectively. Deletion of the HpALG11 gene leads to poor growth and temperature-sensitive lethality, whereas over-expression of HpRft1p can improve growth of the Hpalg11Δ and Hpalg3Δalg11Δ strains. Furthermore, deletion of the HpALG11 gene in the Hpalg3Δ strain resulted in the secretion of glycoproteins with a predicted structure mainly containing trimannosyl core N-linked glycans (Man(3)GlcNAc(2)).

Humanization of Yeasts for Glycan-Type End-Products

“…A more effective biocatalyst system for producing sialylated glycoproteins was established in S. cerevisiae via the deletion of OCH1 and expression of ST more than one decade ago (Shenoy et al, 2021 ). Sialylated O-linked glycans were produced in P. pastoris via the expression of β-1,2-N-acetylglucosaminyltransferase 1 (AST; Song et al, 2010 ).…”

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

“…Here, we focus on the steps and approaches leading to the formation of the simplest complex-type N-glycan GlcNAc 2 Man 3 GlcNAc 2 . Undesired and not well documented side effects of the glycoengineering are often growth defects that can stem from the approach chosen for generation of the glycan acceptor and from the introduction of the required glycan processing enzymes into the Golgi apparatus ( Jacobs et al, 2009 ; Song et al, 2010 ; Parsaie Nasab et al, 2013 ).…”

“…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 ).…”

“…Due to the strong modification of the LLO biosynthesis, measures to compensate for the resulting hypoglycosylation phenotypes are required. 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 ).…”