Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation

We have studied the effects of overexpression and secretion of a homologous model glycoprotein, glucoamylase (GAM-1), on glycosylation in a single gen e copy wild-type parent and multiple gene copy transformants of Aspergillus niger. In batch culture the B36 strain, which possess 80 additional copies of the GAM glaA gene, secreted about 5-8-fold more protein and GAM-1 than the parent strain (N402). A comparison of the glycosylation of GAM-1 secret ed by the parent strain with that secreted by the multiple copy and hyper-s ecreting B36 strain showed that both the N-linked and O-linked glycan compo sition was very similar. Short oligomannose N-linked glycans were found (Ma n-(7-8)GlcNAc(2)). O-Linked glycans were comprised of short (1-3 residues) oligosaccharide chains of mannose and galactose, Evidence is presented that this galactose is present in the novel galactofuranose conformation. This glycan composition of GAM-1 differed from that of a commercially available (A. niger.) GAM source. Microsomes prepared from the mycelium showed a 2-3- fold co-ordinated increase in the activity of the dolichol phosphate:glycos yltransferases. Similar results were obtained from strains B1 (20 copies of glaA) and N402 when grown at a low dilution rate in a chemostat, although both the levels of GAM secretion and the activities of the dolichol phospha te :glycosyltransferases were lower than found in batch culture. These data suggest that A. niger is capable of secreting large amounts of a single gl ycoprotein combined with higher activity levels of the dolichol phosphate:g lycosyltransferases without an increase in the heterogeneity of the glycan structures. Thus, from a biotechnological viewpoint, protein glycosylation may not be a bottleneck to enhanced glycoprotein production using A. niger. (C) 1999 Elsevier Science B.V. All rights reserved.