A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae |
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Authors: | Satoshi Suzuki Sawaki Tada Hiroko Taketani Mayumi Matsushita Ken-Ichi Kusumoto Masanori Sugiyama |
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Affiliation: | a Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan b National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan |
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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 200 μg/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 30 μg 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. |
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Keywords: | Aspergillus oryzae ATP-binding cassette (ABC) transporter Bleomycin Chlorpromazine Drug resistance marker |
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