Marleen Maras

Filamentous fungi as production organisms for glycoproteins of bio-medical interest

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20–40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-d-mannosidase

Abstract A cDNA encoding 1,2-α- d -mannosidase mds1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56u2008266 Da and shows high similarity to the amino acid sequences of 1,2-α- d -mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris . Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae α-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analysed. The enzyme was characterized as a class-I mannosidase.

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei

The human N‐acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT‐D‐80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N‐glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

Modification of the protein glycosylation pathway in the methylotrophic yeast Pichia pastoris.

Abstractα-1,2-Mannosidase from Trichoderma reesei was used to modify the N-linked glycosylation pathway of the methylotrophic yeast Pichia pastoris. Expression of foreign influenza glycoproteins with more extensively processed N-linked oligosaccharides was observed when α-1,2-mannosidase was secreted in the culture medium. However, intracellular removal of mannose residues may stimulate mannosyltransferase activity and leads to hyperglycosylation. [3H]Mannose suicide selection or high concentrations of orthovanadate, commonly used to isolate glycosylation mutants of Saccharomyces cerevisiae, had no profound effect on Pichia pastoris.