Gregg L.F. Wallis

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.

An extracellular β-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis

Aspergillus niger produces an extracellular beta-galactofuranosidase, which can specifically hydrolyse beta-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when beta-galactofuranosidase is absent, suggesting in situ beta-galactofuranosidic hydrolysis. The beta-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS-PAGE) with activity against beta-linked Galf residues, with a Km of 4 mM against p-nitrophenyl-beta-D-galactofuranoside and a pH optimum of 3-4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-beta-D- and -alpha-D-galactopyranose, and alpha-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.

Investigation of the glycosyltransferase enzymes involved in the initial stages of the N-linked protein glycosylation pathway in Aspergillus niger

A protocol has been developed to produce functional microsomes from mycelium of Aspergillus niger. Using these preparations, radioactive biochemical assays have been designed and optimised to measure the in vitro activities of three of the enzymes of the N-linked dolichol phosphate (Dol-P) glycosylation pathway: UDP-N-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), Dol-P mannose synthase (DPMS) and Dol-P glucose synthase (DPGS). Exogenous Dol-P and Triton X-100 are essential for in vitro activity. All three Dol-P:glycosyl transferases had alkaline pH optima and activity rapidly decreased below neutrality. Characterisation of the glycolipid products by anion-exchange chromatography and TLC showed that they were Dol-P-P-GlcNAc, Dol-P-Glc and Dol-P-Man for GPT, DPGS and DPMS, respectively. The antibiotic tunicamycin completely inhibited GPT activity at a concentration of 100-200 ng ml(-1) and an IC50 of 40 ng ml(-1), but had little effect on the other two enzymes. The ratio of the activity of the three enzymes to each other suggested that DPMS may be involved in other cellular activities and is probably under different control mechanisms than the other two.

Protein secretion by fungi

Abstract Protein secretion is a vital process in fungi. For many, the secretion of hydrolytic enzymes provides a crucial step in their nutrition in nature. However, in recent years the list of different types of secreted proteins that have been discovered has extended significantly. These have been shown to have a diversity of functions including toxic molecule transport and control of desiccation. The majority of secreted proteins are glycosylated and our understanding of this aspect of fungal biochemistry has also extended in recent years. This review addresses the process of protein secretion from the cytological, biochemical and genetical standpoints. Advances in technology in many areas of scientific approach have enabled a better and growing understanding of this important process in fungi.