Transformation of aspergillus oryzae through plasmid-mediated complementation of the methionine-auxotrophic mutation

A methionine-auxotrophic mutant (Met^‒) of Aspergillus oryzae was transformed to prototrophy (Met^‒) with plasmid pKA5-l carrying a 3.5 Kbp genomic DNA fragment of A. oryzae through treatment of spheroplasts with polyethylene glycol 4000 and CaCl₂. The highest transformation frequency obtained was 32 transformants per pg DNA. We recovered the transforming plasmid from the Met⁺ transformant cell, showing that this plasmid was derived from the original plasmid, pKA5-l. The mitotic stability of the Met⁺ transformant was about 70% after growth without selective pressure; the transforming DNA was lost in the Met^‒ clone. These findings suggest that the transforming plasmid, pKA5-l, exists in the transformant cell as a self-replicating circular DNA. The gene in the 3.5 Kbp DNA fragment, which complements the methionine-auxotrophic mutation, can be used as a selectable marker to construct efficient plasmid vectors for A. oryzae.