6 research outputs found

    AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus <i>Aspergillus oryzae</i>

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    <p>Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus <i>Aspergillus oryzae</i> AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast <i>Pichia pastoris</i>. Deletion of <i>Aoatg26</i> caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient <i>A. oryzae</i> strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the <i>Aoatg26</i> deletion strain, indicating that autophagic process is impaired. Indeed, the <i>Aoatg26</i> deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in <i>A. oryzae</i>, which may physiologically contribute to the differentiation in filamentous fungi.</p> <p>Involvement of AoAtg26 in autophagic organelle degradation.</p

    Conidiation in autophagy gene-conditional expression strains.

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    <p>(A) Images of the <i>Aoatg</i> conditional expression strains after growth on PD agar plates supplemented with or without thiamine for 4 days at 30°C. (B) The number of conidia formed per plate are shown for each strain under the indicated conditions. Three experiments were performed, and the values of the average and standard deviations are represented (*<i>p</i><0.01, Student’s <i>t</i> test).</p

    Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus <i>Aspergillus oryzae</i>

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    <div><p><i>Aspergillus oryzae</i> has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, <i>A. oryzae</i> often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (<i>Aoatg</i>) gene disruptants of <i>A. oryzae</i>. We transformed <i>A. oryzae</i> gene disruptants of <i>Aoatg1</i>, <i>Aoatg13</i>, <i>Aoatg4</i>, <i>Aoatg8</i>, or <i>Aoatg15</i>, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the <i>Aoatg</i> gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed <i>Aoatg</i> gene-conditional expression strains in which the promoter region of the <i>Aoatg</i> gene was replaced with the thiamine-controllable <i>thiA</i> promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the <i>Aoatg</i> gene disruptants. Consequently, we succeeded in the construction of <i>A. oryzae</i> strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in <i>A. oryzae</i>.</p></div

    Extracellular bovine chymosin (CHY) production by <i>A.oryzae</i> autophagy gene-conditional expression strains.

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    <p>(A) Approximately 2×10<sup>5</sup> conidia of the control (SlD-AKC1), SlD-PtA1-AKC, SlD-PtA4-AKC, SlD-PtA8-AKC, and SlDPtA15-AKC strains expressing CHY were inoculated into 20 ml 5×DPY medium (pH 5.5) supplemented with and without thiamine. CHY activities in the culture supernatant were measured after 4 days of growth at 30°C. Five experiments were performed, and the values of the average and standard deviations are represented (*<i>p</i><0.01, Student’s <i>t</i> test). (B) Western blot analysis of the culture supernatant of the CHY-expressing strains. Mature CHY bands of 35.4 kDa were detected using an anti-CHY antibody.</p

    Extracellular bovine chymosin (CHY) production by <i>A. oryzae</i> autophagy gene disruptants.

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    <p>Approximately 2×10<sup>5</sup> conidia of each strain were inoculated into 20 ml 5×DPY medium (pH 5.5) and the CHY activity in the culture supernatant was measured daily after 3–6 days of growth at 30°C. Three experiments were performed, and the values of the average and standard deviations are represented (*<i>p</i><0.01, Student’s <i>t</i> test).</p
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