Aspergillus niger is used as a host for heterologous protein production, however, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a similar mechanism to other eukaryotes with proteins destined for secretion being directed to the hyphal tip. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A.niger glucoamylase (GLA:499) in order to monitor protein trafficking through the secretory pathway. Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A.nigergenome. Confocal and fluorescence microscopy has shown that GLA::sGFP fusion protein is fluorescent in A.niger and appeared to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence was apparent and immunogold labelling of sGFP confirmed that sGFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::sGFP was only detected in culture filtrates of young mycelia grown in a soya milk medium. Current work is focusing on the disruption the secretory pathway by secretion inhibitors.
Fungal Genet. Newsl. 46 (Supl):
Full conference title:
Fungal Genetics Conference 20th
- Fungal Genetics Conference 20th (1999)