Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae

Mari A. Piirainen, Heidi Salminen, Alexander D. Frey*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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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.

Original languageEnglish
Pages (from-to)301-315
Number of pages15
JournalApplied Microbiology and Biotechnology
Volume106
Issue number1
Early online date15 Dec 2021
DOIs
Publication statusPublished - Jan 2022
MoE publication typeA1 Journal article-refereed

Keywords

  • Galactosyltransferase
  • Glucosamine
  • Glycoengineering
  • UDP-glucose 4-epimerase
  • Yeast

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