title: A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae.
abstract: Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Hiroshima University, Kasumi, Minami-ku, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Sawaki
authors.initials: S
authors.lastname: Tada
authors.forename: Mari
authors.initials: M
authors.lastname: Fukuoka
authors.forename: Hiroko
authors.initials: H
authors.lastname: Taketani
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Tsukakoshi
authors.forename: Mayumi
authors.initials: M
authors.lastname: Matsushita
authors.forename: Kosuke
authors.initials: K
authors.lastname: Oda
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kusumoto
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kashiwagi
authors.forename: Masanori
authors.initials: M
authors.lastname: Sugiyama