Background: The filamentous fungus Aspergillus oryzae is widely used for secondary metabolite production by heterologous expression; thus, a wide variety of promoter tools is necessary to broaden the application of this species. Here we built a procedure to survey A. flavus genes constitutively highly expressed in 83 transcriptome datasets obtained under various conditions affecting secondary metabolite production, to find promoters useful for heterologous expression of genes in A. oryzae. Results:To test the ability of the promoters of the top 6 genes to induce production of a fungal secondary metabolite, ustiloxin B, we inserted the promoters before the start codon of ustR, which encodes the transcription factor of the gene cluster responsible for ustiloxin B biosynthesis, in A. oryzae. Four of the 6 promoters induced ustiloxin B production in all tested media (solid maize, liquid V8 and PDB media), and also ustR expression. Two of the 4 promoters were those of tef1 and gpdA, which are well characterized in A. oryzae and A. nidulans, respectively, whereas the other two, those of AFLA_030930 and AFLA_113120, are newly reported here and show activities comparable to that of the gpdA promoter with respect to induction of gene expression and ustiloxin B production. Conclusion:We newly reported two sequences as promoter tools for secondary metabolite production in A. oryzae. Our results demonstrate that our simple strategy of surveying for constitutively highly expressed genes in large-scale transcriptome datasets is useful for finding promoter sequences that can be used as heterologous expression tools in A. oryzae.

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