title: Modulating endoplasmic reticulum-Golgi cargo receptors for improving secretion of carrier-fused heterologous proteins in the filamentous fungus Aspergillus oryzae.
abstract: 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 α-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 α-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.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
authors.forename: Huy-Dung
authors.initials: HD
authors.lastname: Hoang
authors.affiliation: Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Maruyama
authors.affiliation: Department of Biotechnology, The University of Tokyo, Tokyo, Japan akitamo@mail.ecc.u-tokyo.ac.jp.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins