Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhBcxr) was further examined for possible use as a transformant selection marker. After transformation with AosdhBcxr, carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhBcxr gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhBcxr functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.