MINI-REVIEW Engineering of glycosylation in yeast and other fungi: current state and perspectives Karen De Pourcq & Kristof De Schutter & Nico Callewaert Received: 2 April 2010 / Revised: 8 June 2010 / Accepted: 8 June 2010 / Published online: 29 June 2010 # Springer-Verlag 2010 Abstract With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifica- tions. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’ s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi. Keywords N-glycosylation engineering . O-glycosylation engineering . Yeast . Fungi . Biopharmaceuticals . Recombinant proteins Introduction The use of recombinant proteins as biopharmaceuticals has been increasing, and high performance expression systems are needed to meet the demands. Most therapeutic proteins require glycosylation to ensure proper folding, function, and activity. Therefore, they cannot be produced in standard prokaryotic expression systems, which lack proper glycosyla- tion machinery. Since glycosylation and other post-translational modifications are essential for these therapeutic glycoproteins, most of them are produced in mammalian cells. However, due to the potential spread of infectious diseases through the use of contaminated protein products and potential shortages in bovine serum supply for large-scale production, there is a growing shift to serum-free expression systems. In particular, yeasts and fungi are being developed as excellent alternative protein expression systems. Yeast and fungi have a more rigid cell wall compared to mammalian and insect cells, which makes them more resistant to shear stress and allows them to grow to high cell densities, which facilitates industrial scale up. In addition, fungal expression systems can grow in simple, chemically defined media, which makes the production costs much lower than in mammalian and insect cells. This, together with the ease of genetic manipulation of microbial organisms and the availability of established molecular tools and know-how, has made yeasts and fungi the expression system of choice when manufacturing costs, speed, and yield are of great importance. Moreover, as eukaryotic organisms, yeast and fungi are able to perform post-translational modifications, such as N- and O- glycosylation, disulfide bond formation, and oligomerization, which are often essential for protein quality and functionality. However, protein glycosylation in microorganisms can be quite different from that in mammalian cells. N-glycans in yeast, for example, are of a heterogeneous high-mannose type. K. De Pourcq : K. De Schutter : N. Callewaert (*) Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, 9052, Ghent-Zwijnaarde, Belgium e-mail: nico.callewaert@dmbr.vib-UGent.be K. De Pourcq : K. De Schutter Unit for Molecular Glycobiology, Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, 9052, Ghent, Belgium N. Callewaert Unit for Molecular Glycobiology, Department of Biochemistry and Microbiology, Ghent University, K.L.-Ledeganckstraat 35, 9000, Ghent, Belgium Appl Microbiol Biotechnol (2010) 87:1617–1631 DOI 10.1007/s00253-010-2721-1