Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae.
Appl Microbiol Biotechnol
; 106(1): 301-315, 2022 Jan.
Article
em En
| MEDLINE
| ID: mdl-34910238
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.
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MEDLINE
Assunto principal:
Schizosaccharomyces
/
Proteínas de Saccharomyces cerevisiae
Limite:
Humans
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article