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 gene 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 secreted by the parent strain with that secreted by the multiple copy and hyper-secreting B36 strain showed that both the N-linked and O-linked glycan composition was very similar. Short oligomannose N-linked glycans were found (Man(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:glycosyltransferases. 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 phosphate:glycosyltransferases were lower than found in batch culture. These data suggest that A. niger is capable of secreting large amounts of a single glycoprotein combined with higher activity levels of the dolichol phosphate:glycosyltransferases 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.
Wallis
G L
GL
School of Biomedical Science (Biochemistry), The Medical School, Queens Medical Centre, The University of Nottingham, Nottingham, UK.
Swift
R J
RJ
Hemming
F W
FW
Trinci
A P
AP
Peberdy
J F
JF
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005964:Glucosyltransferases / Q000378:metabolism; D006031:Glycosylation; D011134:Polysaccharides / Q000032:analysis; D013997:Time Factors
D011134:Polysaccharides; D005964:Glucosyltransferases; C054676:dolichol phosphate glucose-dolichol diphosphate oligosaccharide glucosyltransferase; D005087:Glucan 1,4-alpha-Glucosidase