The Studies On Siderophore Biosynthesis And Regulation Of Marine Yeast Aureobasidium Pullulans HN6.2

Aureobasidium pullulans HN6.2, which was isolated from marine environment, could produce high amount of hydroxymate siderophore fusigen. Under optimized condition, siderophore production of this yeast could reach up to0.44mM and would decrease significantly if supplementing with Fe3+. Additionally, the siderophore from this yeast possessed an obvious ability to suppress the growth of marine pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. However, the siderophore biosynthesis and regulation of this yeast at molecular levels still remain unknown.The biosynthesis of fusigen commences with the N5-hydroxylation of L-ornithine catalyzed by an L-ornithine-N5-monooxygenase, producing N5-hydroxyl-L-ornithine which is the basic unit to form fusigen siderophores. The assembly to the final siderophore is catalyzed by non-ribosomal peptide synthetases (NRPSs). Therefore, L-ornithine-N5-monooxygenase is involved in the first step of the fungal siderophores biosynthesis.In order to study the functional genes involved in the siderophore biosynthesis and regulation of this yeast, a gene knock-out module was constructed. This module consisted of TEF promoter, hygromycin B phosphotransferase gene (HPT gene) and specific terminator (polyA) and was nominated as pGDMIAP-1. After connecting the5’-homologous recombination fragment (5’-arm) and3’-homologous recombination fragment (3’-arm) from the alkaline protease gene of strain HN6.2to the both ends of pGDMIAP-1, the alkaline protease gene knock-out vector was obtained. After the transformation of this vector into strain HN6.2, the selected disruptant exhibited a significant decrease in alkaline protease activity, indicating that the gene knock-out module could function properly in strain HN6.2and the construction of gene knock-out vector was successful.In this study, the L-ornithine-N5-monooxygenase structural gene (SidA gene, accession number:FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast A. pullulans HN6.2by inverse PCR and RT-PCR. An open reading frame of1,461bp encoding a486amino acid protein (isoelectric point:7.79) with calculated molecular weight of55.4kDa was characterized. The promoter of the gene (intronless) was located from-28to-77and had one TATA box. The up-stream of the gene also contained two GATA boxes which were the binding sites of transcriptional repressor. The SidA gene in A. pullulans HN6.2was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6(ΔsidA) did not synthesize both intracellular and extracellular siderophore so that t could not inhibit growth of the pathogenic bacteria Ⅴ. anguillarum and Ⅴ. parahaemolyticus. The disruptant S6did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with10μM Fe3+and Fe2+. H2O2in the medium was more toxic to the disruptant S6than to its wild type HN6.2. Thus, we infer that the siderophore produced by the marine-derived A. pullulans HN6.2can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells.At the same time, expression of these genes and enzymes related to the siderophore biosynthesis must be under tight and subtle regulatory. One major iron regulator which positively control the siderophore biosynthesis acts via transcriptional activation and the opposite major iron regulator which negatively regulate siderophore biosynthesis is a highly conserved GATA-type transcriptional repressor containing the GATA-type zinc fingers that bind to promoters of siderophore biosynthetic gene. So in the present study, the GATA-type transcriptional repressor structural gene SRE1gene was isolated from both the genomic DNA and cDNA of the marine yeast A. pullulans HN6.2by inverse PCR and RT-PCR. An open reading frame (ORF) of1002bp encoding a334amino acid protein (isoelectric point:8.6) with a calculated molecular weight of35.1kDa was characterized. The corresponding gene had one single intron of51bp, and in its promoter two putative5’-HGATAR-3’ sequences could be recognized. The deduced protein from the cloned gene contained two conserved zinc-finger domains [Cys-(X2)-Cys-(X17)-Cys-(X2)-Cys)], nine sequences of Ser(Thr)-Pro-X-X which is characteristics of the regulator, and one cysteine-rich central domain which is located between the two zinc fingers. The SRE1gene in A. pullulans HN6.2was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into the ORF of the SRE1gene using homologous recombination. Of all the disruptants obtained, one strain R6(Asre1) still synthesized both intracellular and extracellular siderophore in the presence of Fe3+and expression of the SidA gene encoding L-ornithine N5-oxygenase in the disruptant R6was also partially derepressed in the presence of Fe3+. The disruptant R6could grow in brown-colored colonies on iron-repleted medium. In contrast, A. pullulans HN6.2could not grow in the iron-repleted medium with high concentration of1.5mM and2.0mM Fe3+. Because the disruptant R6could produce high amount of siderophore in iron-replete medium, the antibacterial activity of the supernatant of disruptant R6obtained from this medium was still obvious.This is the first time to carry out the research work on biosynthesis and regulation of the siderophore in marine yeasts.