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Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes ∼3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

Therapeutic glycoprotein production in the widely used expression host Pichia pastorisis hampered by the differences in the protein‐linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human‐compatible N‐glycans in this organism is the conversion of the yeast‐type high‐mannose glycans to mammalian‐type high‐mannose and/or complex glycans. In this perspective, we have co‐expressed an endoplasmic reticulum‐targeted Trichoderma reesei1,2‐α‐D‐mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans‐sialidase. Analysis of the N‐glycans of the two purified proteins showed a >85% decrease in the number of α‐1,2‐linked mannose residues. Moreover, the human‐type high‐mannose oligosaccharide Man5GlcNAc2was the major N‐glycan of the glyco‐engineered trans‐sialidase, indicating that N‐glycan engineering can be effectively accomplished in P. pastoris.

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

Therapeutic glycoprotein production in the widely used expression host Pichia pastorisis hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei1,2-α- D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of α-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man 5GlcNAc 2was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.

The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N- and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: α-(1→2)-mannosidase, α-(1→3)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor–enriched media, where the pH is closer to the pH optimum (5–6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by α-(1→2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.