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-Nacetylglucosamine 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.

Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

“…One of the main limitations of producing recombinant pharmaceuticals in yeast is the different N -glycan pattern compared to mammalian expression platforms [ 2 ]. However, producing correctly folded and glycosylated proteins for therapeutic applications in S. cerevisiae is about to become possible with the advancements of glycoengineered strains [ 3 , 4 ]. With further optimization of the host and the production process, the yeast has great potential to become a viable platform to produce competitive yields of complex, functional mammalian target proteins, such as antibodies.…”

Investigating the role of ERAD on antibody processing in glycoengineeredSaccharomyces cerevisiae

“…For deglycosylation, the sample was denatured and treated with PNGase F overnight (New England Biolabs, Ipswich, MA, USA) according to manufacturer's instructions. The released glycans were purified using C18 and graphitized carbon columns as described earlier (Piirainen et al 2016).…”

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

“…Mammalian glycosyltransferases also utilize nucleotide sugars including UDP-GlcNAc and UDP-galactose not present in the Golgi apparatus of most yeasts and improving their availability has often been required to obtain efficient conversions by the mammalian glycosyltransferases. Expression of a UDP-GlcNAc transporter from Kluyveromyces lactis (Yea4, alternatively called Mnn2-2) has increased the amount of hybrid and complex-type glycans in S. cerevisiae and other yeasts (Choi et al 2003 ; Bobrowicz et al 2004 ; Wang et al 2013 ; Piirainen et al 2016 ). In addition, incorporation of a UDP-glucose 4-epimerase from Schizosaccharomyces pombe into a GalT fusion protein has provided efficient N-glycan galactosylation (Bobrowicz et al 2004 ; Jacobs et al 2009 ; Wang et al 2013 ).…”