Transformation of Aspergillus oryzae RIB40 with the bleomycin acetyltransferase-encoding gene as a selection marker

Satoshi Suzuki1, Mari Fukuoka1, Hiroko Taketani1, Sawaki Tada1, Mayumi Matsushita1, Ken-Ichi Kusumoto1, Yutaka Kashiwagi1, Masanori Sugiyama2

Author address: 

1National Food Research Institute, Tsukuba, Ibaraki, Japan, 2Hiroshima University, Hiroshima, Hiroshima, Japan


Introduction: Aspergillus oryzae is one of the most important fungi in Japanese traditional fermentation industry. Since this microorganism also produces many-food processing enzymes, it is significant to establish a host-vector system for A. oryzae. In various filamentous fungi, the genes, which confer resistance to hygromycin B, aureobasidin, and G418, have been used as genetic markers for the gene manipulation. However, A. oryzae is resistant to these antibiotics. We notice that high concentration of bleomycin inhibits the growth of A. oryzae RIB40 which is known as a genome reading strain. We have recently found that some agents, such as tritonX100, increase the bleomycin-susceptibility of A. oryzae (data not shown). In the present study, we developed a transformation system for A. oryzae RIB40 by using bleomycin-resistance gene as a selective marker. Methods: The codon of the bleomycin N-acetyltransferase gene from Streptomyces verticillus were optimized according to the codon usage of the fungus and inserted into the plasmid pPTRI under control of the histone H2B promoter. The resulting chimeric plasmid was introduced into the protoplasts from A. oryzae RIB40 by the polyethylene glycol method. The transformants were selected on the maltextract-peptone medium containing 27 microgram/ml of bleomycin and bleomycin potentiation agents. The integration of the plasmid into genomic DNA of transformants was confirmed by the colony PCR method using FTA card and the Southern blot analysis. Results: We successfully selected the transformants, which exhibit bleomycin resistance, on the agar plate. The background outgrowth of non-transformants was clearly suppressed by bleomycin. The transformants were inoculated on the non-selective PDA plate and allowed to grow. The colony PCR analysis shows that the growing tip of colony inoculated on non-selective medium retains the DNA fragment carrying the bleomycin N-acetyltransferase gene. Discussion: In the present study, we established the transformation system for A. oryzae by using the Streptomyces bleomycin N-acetyltransferase gene. We expect that the established system contribute to many research for A. oryzae

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Full conference title: 

    • ECFG 9th (2008)