title: Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae.
abstract: Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously produced proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (DeltaligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (DeltatppA DeltapepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous protein by A. oryzae.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Jaewoo
authors.initials: J
authors.lastname: Yoon
authors.forename: Shinya
authors.initials: S
authors.lastname: Kimura
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Maruyama
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
mesh_terms: D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D012085:Chymosin / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D005656:Fungal Proteins; D010447:Peptide Hydrolases; D012085:Chymosin