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Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae
Ist Teil von
Applied microbiology and biotechnology, 2022-01, Vol.106 (1), p.301-315
Ort / Verlag
Berlin/Heidelberg: Springer Berlin Heidelberg
Erscheinungsjahr
2022
Link zum Volltext
Quelle
SpringerLink
Beschreibungen/Notizen
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
Δ
alg3
Δ
alg11
strain where a truncated Man
3
GlcNAc
2
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.