<i>In vivo</i>Visualization of the Distribution of a Secretory Protein in<i>Aspergillus oryzae</i>Hyphae Using the RntA-EGFP Fusion Protein

Masai, Kumiko; Maruyama, Jun‐ichi; Nakajima, Harushi; Kitamoto, Katsuhiko

doi:10.1271/bbb.67.455

Cited by 35 publications

(31 citation statements)

References 13 publications

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“…3C and D). Moreover, these observations were consistent with the known septal localization patterns of secretory hydrolytic enzymes fused with (E)GFP, such as the RNase of A. oryzae (27) and the glucoamylase of A. niger (9). The dispersed, round structures observed in the ⌬Aovps10 strain were attributed to accumulation of the fusion protein at unknown organelles ( Fig.…”

Section: Resultssupporting

confidence: 86%

“…The Vps10p receptor has also been implicated in the targeting and delivery of several recombinant proteins from the late-Golgi compartments to vacuoles for degradation by the vacuolar protease complex (11,12). In our previous work, secreted protein RNase T 1 (RntA) fused with enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in the vacuoles, as well as in the hyphal tips (27), indicating that a portion of the expressed heterologous proteins was presumably transported into the vacuoles for degradation. These observations suggest that for the improvement of heterologous protein production in A. oryzae, it is necessary to elucidate the machinery which mediates protein trafficking between the Golgi apparatus and vacuoles.…”

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confidence: 99%

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Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Yoon

Aishan

Maruyama

et al. 2010

Appl Environ Microbiol

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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, ⌬Aovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the ⌬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.

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Section: Resultssupporting

confidence: 86%

mentioning

confidence: 99%

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Yoon

Aishan

Maruyama

et al. 2010

Appl Environ Microbiol

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“…Thus, we hypothesized that hfm-21 secretes CPY-EGFP via a vesicle-mediated, conventional secretory pathway similar to the one used for extracellular enzymes. Unlike glucoamylase-green fluorescent protein fluorescence (9) and RNase T1-EGFP fluorescence (12), however, CPY-EGFP fluorescence was not observed at the septa. Therefore, the vesicles transporting CPY-EGFP or ␣-amylase may be distinguishable from those transporting glucoamylase or RNase T1 in this mutant.…”

Section: Discussionmentioning

confidence: 81%

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Ohneda

Arioka

Kitamoto

2005

Appl Environ Microbiol

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The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10-to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

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“…3). However, the Spitzenkörper where the secretory proteins accumulate (Gordon et al, 2000;Harris et al, 2005;Hayakawa et al, 2011;Kimura et al, 2010;Masai et al, 2003) could not be observed at the hyphal tips.…”

Section: Cloning Of Aosedd and Localization Analysis Of Aosedd-egfpmentioning

confidence: 90%

Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae

Zhu

Nemoto

Yoon

et al. 2012

J. Gen. Appl. Microbiol.

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In vivoVisualization of the Distribution of a Secretory Protein inAspergillus oryzaeHyphae Using the RntA-EGFP Fusion Protein

Cited by 35 publications

References 13 publications

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae

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