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.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Aspergillus oryzae/genetics , Bleomycin/pharmacology , Drug Resistance, Fungal/genetics , Transformation, Genetic , Acyltransferases/genetics , Aspergillus oryzae/drug effects , Culture Media/chemistry , Culture Media/pharmacology , Gene Knockout Techniques , Gene Transfer Techniques , Selection, GeneticABSTRACT
High-throughput genotyping of Aspergillus oryzae was achieved using an FTA card for the extraction of a genomic DNA template for polymerase chain reaction from a fungal colony growing on an agar plate. This method was then applied to detect other fungal species from agar slants and food materials. This method offers a convenient tool for the genotyping of filamentous fungi without using an organic solvent or specialized equipment.
Subject(s)
Aspergillus oryzae/genetics , Aspergillus oryzae/isolation & purification , Chromosome Mapping/methods , DNA, Fungal/genetics , Genome, Fungal/genetics , Polymerase Chain Reaction/methods , GenotypeABSTRACT
High-throughput screening of enhanced green fluorescent protein (EGFP) gene-trap transformants of filamentous fungus was achieved for the first time using an image analyzer to measure their fluorescent intensity. For quantitative analysis of EGFP fluorescent intensity per unit cell mass, we developed a method for measurement of cell mass using the fluorescent dye SP-Dil. This method offers an effective and convenient tool for screening transformants expressing fluorescent protein as a reporter, and for quantitative analysis of fungal cell mass at jig levels.
