Abstract
N-glycosylation is an important feature of therapeutic
and other industrially relevant proteins, and engineering
of the N-glycosylation pathway provides opportunities for
developing alternative, non-mammalian glycoprotein
expression systems. Among yeasts, Saccharomyces
cerevisiae is the most established host organism used in
therapeutic protein production and therefore an
interesting host for glycoengineering. In this work, we
present further improvements in the humanization of the
N-glycans in a recently developed S. cerevisiae strain.
In this strain, a tailored trimannosyl lipid-linked
oligosaccharide is formed and transferred to the protein,
followed by complex-type glycan formation by Golgi
apparatus-targeted human N-acetylglucosamine
transferases. We improved the glycan pattern of the
glycoengineered strain both in terms of glycoform
homogeneity and the efficiency of complex-type
glycosylation. Most of the interfering structures present
in the glycoengineered strain were eliminated by deletion
of the MNN1 gene. The relative abundance of the
complex-type target glycan was increased by the
expression of a UDP-N-acetylglucosamine transporter from
Kluyveromyces lactis, indicating that the import of
UDP-N-acetylglucosamine into the Golgi apparatus is a
limiting factor for efficient complex-type
N-glycosylation in S. cerevisiae. By a combination of the
MNN1 deletion and the expression of a
UDP-N-acetylglucosamine transporter, a strain forming
complex-type glycans with a significantly improved
homogeneity was obtained. Our results represent a further
step towards obtaining humanized glycoproteins with a
high homogeneity in S. cerevisiae.
Original language | English |
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Pages (from-to) | 189-199 |
Journal | Glycoconjugate Journal |
Volume | 33 |
Issue number | 2 |
DOIs | |
Publication status | Published - 2016 |
MoE publication type | A1 Journal article-refereed |
Keywords
- Glycoengineering
- Glycosylation efficiency
- MNN1
- N-glycosylation
- UDP-GlcNAc transporter
- Yeast