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

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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.

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Acknowledgments

The work was supported by Aalto University School of Chemical Technology. MA Piirainen is a recipient of a doctoral study grant from the School of Chemical Technology, Aalto University.

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Authors and Affiliations

  1. Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1, 02150, Espoo, Finland

    Mari A. Piirainen, Jorg C. de Ruijter & Alexander D. Frey

  2. VTT Technical research centre of Finland, Espoo, Finland

    Harry Boer

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  1. Mari A. Piirainen
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Correspondence to Alexander D. Frey.

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Piirainen, M.A., Boer, H., de Ruijter, J.C. et al. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae . Glycoconj J 33, 189–199 (2016). https://doi.org/10.1007/s10719-016-9656-4

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  • DOI: https://doi.org/10.1007/s10719-016-9656-4

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