A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae

Mari Piirainen, Harry Boer, Jorg C. de Ruijter, Alexander D. Frey

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

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 languageEnglish
Pages (from-to)189-199
JournalGlycoconjugate Journal
Volume33
Issue number2
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

Fingerprint

Glycosylation
Yeast
Polysaccharides
Saccharomyces cerevisiae
Golgi Apparatus
N-Acetylglucosaminyltransferases
Glycoproteins
Uridine Diphosphate N-Acetylglucosamine
Kluyveromyces
Protein Engineering
Proteins
Uridine Diphosphate
Acetylglucosamine
Gene Deletion
Genes
Yeasts
Therapeutics
UDP-N-acetylglucosamine transporter

Keywords

  • Glycoengineering
  • Glycosylation efficiency
  • MNN1
  • N-glycosylation
  • UDP-GlcNAc transporter
  • Yeast

Cite this

Piirainen, Mari ; Boer, Harry ; de Ruijter, Jorg C. ; Frey, Alexander D. / A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae. In: Glycoconjugate Journal. 2016 ; Vol. 33, No. 2. pp. 189-199.
@article{3c01bff90b8843fb8595d8b9c374afdf,
title = "A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae",
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.",
keywords = "Glycoengineering, Glycosylation efficiency, MNN1, N-glycosylation, UDP-GlcNAc transporter, Yeast",
author = "Mari Piirainen and Harry Boer and {de Ruijter}, {Jorg C.} and Frey, {Alexander D.}",
year = "2016",
doi = "10.1007/s10719-016-9656-4",
language = "English",
volume = "33",
pages = "189--199",
journal = "Glycoconjugate Journal",
issn = "0282-0080",
publisher = "Springer",
number = "2",

}

A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae. / Piirainen, Mari; Boer, Harry; de Ruijter, Jorg C.; Frey, Alexander D.

In: Glycoconjugate Journal, Vol. 33, No. 2, 2016, p. 189-199.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae

AU - Piirainen, Mari

AU - Boer, Harry

AU - de Ruijter, Jorg C.

AU - Frey, Alexander D.

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

KW - Glycoengineering

KW - Glycosylation efficiency

KW - MNN1

KW - N-glycosylation

KW - UDP-GlcNAc transporter

KW - Yeast

U2 - 10.1007/s10719-016-9656-4

DO - 10.1007/s10719-016-9656-4

M3 - Article

VL - 33

SP - 189

EP - 199

JO - Glycoconjugate Journal

JF - Glycoconjugate Journal

SN - 0282-0080

IS - 2

ER -