Using media with low water activity, a large numbers of aureobasidium-like black yeasts were isolated from glacial and subglacial ice of three polythermal glaciers from the coastal Arctic environment of Kongsfjorden (Svalbard, Spitsbergen), as well as from adjacent sea water, sea ice and glacial meltwaters. To characterise the genetic variability of Aureobasidium pullulans strains originating from the Arctic and strains originating pan-globally, a multilocus molecular analysis was performed, through rDNA (internal transcribed spacers, partial 28 S rDNA), and partial introns and exons of genes encoding beta-tubulin (TUB), translation elongation factor (EF1alpha) and elongase (ELO). Two globally ubiquitous varieties were distinguished: var. pullulans, occurring particularly in slightly osmotic substrates and in the phyllosphere; and var. melanogenum, mainly isolated from watery habitats. Both varieties were commonly isolated from the sampled Arctic habitats. However, some aureobasidium-like strains from subglacial ice from three different glaciers in Kongsfjorden (Svalbard, Spitsbergen), appeared to represent a new variety of A. pullulans. A strain from dolomitic marble in Namibia was found to belong to yet another variety. No molecular support has as yet been found for the previously described var. aubasidani. A partial elongase-encoding gene was successfully used as a phylogenetic marker at the (infra-)specific level.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding �\-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 �� 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 �� 1.95 g/l pullulan within 132 h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.

Aureobasidium melanogenum HN6.2 is a unique yeast strain who can produce the siderophore of fusigen under iron starvation to guarantee its survival. However, a comprehensive understanding of mechanisms involved in iron acquisition and homeostasis for it is still vacant. In this study, genome sequencing and mining revealed that A. melanogenum HN6.2 strain was the first yeast species that exclusively possessed all the four known mechanisms for the iron acquisition: (i) the siderophore-mediated iron uptake; (ii) reductive iron assimilation; (iii) low-affinity ferrous uptake; and (iv) heme utilization, which suggested its stronger adaptability than Aspergillus fumigatus and Saccharomyces cerevisiae. This HN6.2 strain also employed the vacuolar iron storage for immobilizing the excessive iron to avoid its cellular toxicity. Specially, genome mining indicated that A. melanogenum HN6.2 strain could also synthesize ferricrocin siderophore. Further HPLC and Q-Tof-MS analysis confirmed that the siderophores synthesized by this strain consisted of cyclic fusigen, linear fusigen, ferricrocin, and hydroxyferricrocin and they played parallel roles as both intracellular and extracellular siderophores. Also, the heme utilization for this strain was experimentally verified by the knock-out of heme oxygenase gene. For iron homeostasis, the transcriptome analysis revealed that this strain mainly employed two central regulators of SreA/HapX to tune iron uptake and storage at the transcriptional level. It was also noted that mitogen-activated protein kinase C gene (MpkC) exhibited a transcriptional up-regulation under iron sufficiency, suggesting that it may serve as another factor involved in the repression of siderophore biosynthesis. This is the first genetic blueprint of iron acquisition and homeostasis for A. melanogenum.

The genomic DNA and cDNA encoding �\-l-arabinofuranosidase were cloned from the dimorphic fungus Aureobasidium pullulans ATCC 20524 and sequenced. The open reading frame (2097 bp) of the �\-l-arabinofuranosidase gene abfB was interrupted by five introns of 49, 49, 50, 65, and 49 bp. The gene encoded a presumed signal peptide of 17 residues and a mature protein of 682 residues with a calculated Mr of 74,230 Da and a theoretical isoelectric point of 4.95. Glu-362 and Glu-440 residues are likely involved in catalytic reactions as an acid/base and a nucleophile, respectively. The protein possessed 15 potential N-glycosylation sites. The deduced amino acid sequence of the abfB gene product was 58% identical to the Penicillium purpurogenum ABF 2, which belongs to the glycoside hydrolase family-51 �\-l-arabinofuranosidase. The abfB cDNA was functionally expressed in the yeast Pichia pastoris. The recombinant enzyme, AbfB, was purified from the culture filtrate, and it appeared as a single band on SDS-PAGE with an apparent Mr of 110 kDa. AbfB showed �\-l-arabinofuranosidase activity of 56.6 U/mg of protein toward p-nitrophenyl (pNP) �\-l-arabinofuranoside at optimal pH 4.5 and 75�XC. The enzyme exhibited apparent Km and Vmax values of 6.27 mM and 78.1 �gmol/mg/min, respectively, for pNP �\-l-arabinofuranoside. The enzyme was highly active on rye arabinoxylan as well as pNP �\-l-arabinofuranoside, but it showed weak activity toward �\-(1��5)-l-arabinobiose, �\-(1��5)-l-arabinotriose, branched l-arabinan, linear �\-(1��5)-l-arabinan, and arabinogalactan.