dffA gene from Aspergillus oryzae encodes L-ornithine N5-oxygenase and is indispensable for deferriferrichrysin biosynthesis

We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N-5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to the Ustilago maydis sid1 protein (Sid1) and Pseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithine N-5-oxygenase. The complete gene sequence shows that the dffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intron, which is a typical intorn length in A. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved in N-hydroxylating enzymes. As well as that of the U. maydis sid1 gene, dffA gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the (x-amylase promoter in A. oryzae, transformants revealed inducible high L-ornithine\ N-5-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae.