Extracellular Production of Neoculin, a Sweet-Tasting Heterodimeric Protein with Taste-Modifying Activity, by
            <i>Aspergillus oryzae</i>

et al. 2010

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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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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

et al. 2010

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“…When the UPR is triggered, the expression of a set of UPR-associated proteins, including molecular chaperones, vesicular traffic components, and ERassociated degradation (ERAD) proteins, is upregulated in an attempt to resolve the ER stress by increasing the folding, transport, and degradation of proteins (13). Constitutive upregulation of UPR by the expression of the activated form of the transcription factor HacA was shown to improve the production of bovine chymosin in Aspergillus niger (14) and the plant taste-modifying protein neoculin in A. oryzae (7). Although these observations suggest that ER stress is a major hindrance for heterologous protein production in filamentous fungi, the underlying cause of the ER stress associated with heterologous protein expression remains unclear.…”

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confidence: 99%

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Hoang

Kitamoto

2015

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Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme ␣-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of ␣-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. Filamentous fungi possess a prominent secretory capacity and, thus, are excellent hosts for recombinant protein production. Numerous approaches and attempts have been made to produce industrially valuable proteins in filamentous fungi, such as Aspergillus and Trichoderma (1). However, higher eukaryotic proteins are generally inefficiently produced and secreted in these fungi compared to the production and secretion of fungal or endogenous proteins. Several bottlenecks in the heterologous protein production process have been identified to date, and a few limiting factors have been overcome by genetically modifying the expression host. In particular, reducing protease activity is necessary to limit the degradation of heterologous proteins, as was demonstrated by the 3-fold increase in the level of heterologous proteins in the culture supernatant of an Aspergillus oryzae strain with 10 protease genes deleted (2). Heterologous protein production by A. oryzae was also effectively improved by the repression of vacuolar protein sorting and autophagy (3, 4). The genetic fusion of a target protein with an endogenous protein carrier is a commonly used strategy to increase he...

“…More recently, the ability of the species to secrete large amounts of proteins has extended its industrial use to include heterologous protein production (28,46,63,64). A. oryzae has no known sexual cycle and has traditionally been classified within the Fungi Imperfecti (the Deuteromycota), or "mitosporic" fungi (35).…”

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confidence: 99%

Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae

Wada

Yamaguchi

et al. 2012

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ABSTRACTThe potential for sexual reproduction inAspergillus oryzaewas assessed by investigating the presence and functionality ofMATgenes. Previous genome studies had identified aMAT1-1gene in the reference strain RIB40. We now report the existence of a complementaryMAT1-2gene and the sequencing of an idiomorphic region fromA. oryzaestrain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of theMAT1-1andMAT1-2genotypes among 180 strains assayed, including industrialtane-kojiisolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in whichMATgenes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both theMAT1-1andMAT1-2genes functionally regulate gene expression inA. oryzaein a mating type-dependent manner, the first such report for a supposedly asexual fungus.MAT1-1expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system inA. oryzae, and prospects for the discovery of a sexual cycle are discussed.