title: Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae.
abstract: Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Yokota
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shiro
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Yasumichi
authors.initials: Y
authors.lastname: Onozaki
authors.affiliation: National Research Institute of Brewing, Higashi-Hiroshima, 739-0046, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Mizutani
authors.affiliation: National Research Institute of Brewing, Higashi-Hiroshima, 739-0046, Japan.
authors.forename: Dararat
authors.initials: D
authors.lastname: Kakizono
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Sakurako
authors.initials: S
authors.lastname: Ichinose
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Shintani
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Gomi
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan. takahiro.shintani.d7@tohoku.ac.jp.
authors.forename: Takahiro
authors.identifier: http://orcid.org/0000-0001-6630-5892
authors.initials: T
authors.lastname: Shintani
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D056811:Unfolded Protein Response; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008361:Mannosidases; D000516:alpha-Amylases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase
keywords: 1,2-α-D-mannosidase; Aspergillus oryzae; Endoplasmic reticulum-associated degradation; Hyperglycosylation; Unfolded protein response; α-Amylase