Filamentous fungi have received attention as a host for heterologous protein production because of their high secretion capability. However, a bottleneck in post-transcriptional processing in the secretory pathway limits protein production yields. The vacuolar protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar proteins. Although it can also target several recombinant and aberrant proteins for the vacuolar degradation, there is limited knowledge of the effect of its disruption on heterologous protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was isolated. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent protein showed that the fusion protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of AoCpyA to vacuoles in A. oryzae. To investigate the extracellular production level of heterologous protein, we constructed an 916;Aovps10 mutant expressing bovine chymosin (CHY). Interestingly, 916;Aovps10 increased the maximum extracellular production by three fold. Western blot analysis of extracellular CHY also demonstrated an improvement in productivity. These results suggest that the AoVps10 plays a role in the regulation of heterologous protein secretion in A. oryzae, and may be involved in vacuolar protein degradation through the Golgi apparatus. This is the first report demonstrating that disruption of a vacuolar protein sorting gene in filamentous fungi leads to enhanced production levels of heterologous protein.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)