DffA Gene fromAspergillus oryzae encodesl-ornithineN5-oxygenase and is indispensable for deferriferrichrysin biosynthesis

Abstract

We identified and analyzed thedffA gene fromAspergillus oryzae which encodesl-ornithineN5-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 anA. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to theUstilago maydis sid1 protein (Sid1) andPseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithineN5-oxygenase. The complete gene sequence shows that thedffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intorn, which is a typical intorn length inA. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and ‘FATGY’ motifs, which are conserved inN-hydroxylating enzymes. As well as that of theU. 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 thedffA gene was expressed under the control of the a-amylase promoter inA. oryzae, transformants revealed inducible highl-ornithineN5-oxygenase activities. In addition, adffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that thedffA gene is indispensable for deferriferrichrysin biosynthesis inA. oryzae.