Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N‐glycan by blocking core oligosaccharide assembly

Abstract

The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.

Glycoengineered Yeast as an Alternative Monoclonal Antibody Discovery and Production Platform

“…Interestingly, the N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain and blocked in the yeastspecific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi -1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man3GlcNAc2), which is the common core backbone of various human-type N-glycans and the glycoform is the same as that of Cerezyme which was achieved through in vitro sequential deglycosylation of complex glycans (Oh et al, 2008). Similar glycoengineering effort was published in a glycoengineered Pichia with the ability of producing glycoprotein carrying similar mannose type of glycan .…”

Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

“…Several yeast and fungal species have been used in the development of secretion hosts producing therapeutic glycoproteins with human complex-type N-glycans (15,16,33). Recently, pioneering work on the engineering of the O-glycosylation pathway expressing human proteins in S. cerevisiae was reported (8).…”

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

“…Yeast cells were routinely grown in YPD medium (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose) or synthetic complete (SC) medium (0.67% yeast nitrogen base without amino acids, 2% glucose, dropout amino acid mixture supplemented with all required amino acids). Hphac1 and Hpire1 deletion mutants were constructed in the H. polymorpha DL1-L background by fusion PCR and in vivo DNA recombination (15), using the primer sets listed in Table 1. PCR fragments were introduced into yeast cells, and the transformants carrying the gene deletion as a result of in vivo DNA recombination were selected first by growth in minimal medium lacking leucine (SC-LEU) and then by PCR screening.…”

“…7B). To confirm the positive effect of HpHac1p on the modulation of glycosylation, we performed the same experiment employing the Hpalg3⌬ mutant strain, which has a defect in the conversion step of Man 5 GlcNAc 2 -dolichyl pyrophosphate (Dol-PP) to Man 6 GlcNAc 2 -Dol-PP in the ER (15). Our previous study reported that the S. cerevisiae CPY protein was hypoglycosylated with a reduced extent of occupancy at N-glycosylation sites in the Hpalg3⌬ mutant (13).…”

“…In recent decades, H. polymorpha has also been used as a host for heterologous protein production (12). In particular, glycoengineered H. polymorpha strains have been developed to produce glycoproteins with human-compatible N-glycans on the basis of information on the host-specific structure and biosynthesis pathway of N-linked glycosylation in H. polymorpha (13)(14)(15). Likewise, a recent study on the identification and functional analysis of protein O-mannosyltransferases (PMTs) in H. polymorpha provided some unique features of PMT proteins in H. polymorpha (16).…”