Development of a Modified Positive Selection Medium That Allows to Isolate Aspergillus oryzae Strains Cured of the Integrated niaD-Based Plasmid

Abstract

The nitrate reductase gene (niaD) is the most frequently utilized as a selectable marker for homologous integration at the niaD locus of Aspergillus oryzae. In this study we developed a method for curing of the niaD-based plasmid integrated on the A. oryzae genome. Positive selection using a modified chlorate medium containing leucine as a nitrogen source enabled efficient isolation of the strains deficient in nitrate assimilation from the niaD ⁺ transformant. PCR analysis of the strains confirmed that the homologously integrated plasmid carrying the h2b-egfp fusion gene was cured by intrachromosomal recombination which was accompanied by the loss of the EGFP-fluorescence.

• Aspergillus oryzae
• curing
• positive selection
• nitrate reductase
• EGFP