Engineering of Yeast Glycoprotein Expression.

Yeasts are valuable hosts for recombinant protein production, as these unicellular eukaryotes are easy to handle, grow rapidly to a high cell density on cost-effective defined media, often offer a high space-time yield, and are able to perform posttranslational modifications. However, a key difference between yeasts and mammalian cells involves the type of glycosylation structures, which hampers the use of yeasts for the production of many biopharmaceuticals. Glycosylation is not only important for the folding process of most recombinant proteins; it has a large impact on pharmacokinetics and pharmacodynamics of the therapeutic proteins as well. Yeasts' hypermannosylated glycosyl structures in some cases can evoke immune responses and lead to rapid clearance of the therapeutic protein from the blood. This chapter highlights the efforts made so far regarding the glyco-engineering of N- and O-type glycosylation, removing or reducing yeast-specific glycans. In some cases, this is combined with the introduction of humanized glycosylation pathways. After many years of patient development to overcome remaining challenges, these efforts have now culminated in effective solutions that should allow yeasts to reclaim the primary position in biopharmaceutical manufacturing that they enjoyed in the early days of biotechnology. Graphical Abstract.

Functional exploration of the GH29 fucosidase family.

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family's large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Customized protein glycosylation to improve biopharmaceutical function and targeting.

For a long time, glycoprotein production has been limited by the inherent properties of production hosts. Glycosylation of biopharmaceuticals has been regarded as a necessary evil, often needed for protein folding or function, but also a source of heterogeneity, complicating downstream processing and product characterization. This has strongly determined the choice of production hosts. Over the last few decades, numerous glycoengineering efforts have helped solving this problem. Moreover, insights from fundamental studies have made it possible to improve therapeutic protein functionality through careful glycoengineering. Here, we will focus on how production host and in vitro glycoengineering approaches allow to design biopharmaceuticals with glycans that impart improved functionality. An important branch of research explores how glycosylation can be tuned to improve pharmacokinetics and reduce glycan heterogeneity of therapeutics. Furthermore, antibody glycoengineering to obtain homogeneous, defined glycan structures has been a major focus. An example of this is the production of Fc glycans without core fucose, exhibiting tremendously improved Antibody-Dependent Cell Cytotoxicity (ADCC). In the last part, glycoforms that allow for improved (subcellular) targeting and cellular uptake, a field that opens possibilities for enzyme replacement therapies and vaccine development, will be highlighted.