Affiliation:
1. Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Abstract
ABSTRACT
Filamentous fungi have received attention as hosts for heterologous protein production because of their high secretion capability and eukaryotic posttranslational modifications. However, despite these positive attributes, a bottleneck in posttranscriptional processing limits protein 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 recombinant and aberrant proteins for 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 cloned and sequenced. 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 vacuolar proteins to vacuoles. To investigate the extracellular production levels of heterologous proteins, Δ
Aovps10
mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the Δ
Aovps10
mutation increased the maximum extracellular production levels of CHY and HLY by 3- and 2.2-fold, respectively. Western blot analysis of extracellular heterologous proteins also demonstrated an improvement in productivity. These results suggest that AoVps10 plays a role in the regulation of heterologous protein secretion in
A. oryzae
and may be involved in the vacuolar protein degradation through the Golgi apparatus.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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