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 pKA5-1 carrying a 3.5Kbp 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 μg DNA. We recovered the transforming plasmid from the Met+ transformant cell, showing that this plasmid was derived from the original plasmid, pKA5-1. 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, pKA5-1, exists in the transformant cell as a self-replicating circular DNA. The gene in the 3.5Kbp DNA fragment, which complements the methionine-auxotrophic mutation, can be used as a selectable marker to construct efficient plasmid vectors for A. oryzae.