Study On Pullulan Production By A Marine Yeast Aureobasidium Melanogenum Strain P16

Pullulan that is a linear a-D-glucan, made mainly of maltotriose repeating units interconnected by a-],6 linkages is the water-soluble homopolysaccharide produced extracellularly by Aureobasidium spp. The regular alternation of a-1.4 and α-1,6 bonds results in two distinctive properties of structural flexibility and enhanced solubility. Pullulan has many potential applications in food, medical, pharmaceutical, agricultural and cosmetic industries.After over 100 strains of Aureobasidium spp. isolated from mangrove system were screened for their ability to produce exopolysaccharide (EPS), it was found that Aureobasidium melanogenium P16 strain among them could produce high level of EPS.120 g/1 sucrose and 3 g/1 yeast extract in the medium were the most suitable for EPS production.65.3±2.1 g/1 EPS and 18.7±0.38 cell mass were produced by P16 strain within 120 h at flask level. During 10-1 batch fermentation, when the medium contained 120 g/1 sucrose,67.4±4.5 g/1 EPS in the culture and 23.01±0.12 g/1 of cell dry weight were obtained within 120 h, leaving 0.078% reducing sugar and 1.1% total sugar in the fermented medium. It should be stressed that during the fermentation, no melanin was observed. After purification, the purified EPS could be actively hydrolyzed by pullulanase. FTIR analysis of the purified EPS showed that the spectra of the purified EPS were consistent with those of the standard pullulan, indicating the chemical structure of the EPS produced by strain P16 was pullulan. This is the first time to report that A. melanogenium P16 strain isolated from mangrove system can produce such high level of pullulan.In order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in A. melanogenum P16 strain. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain E136, one of the transformants, produced 40.92 U/ml of inulinase activity while its wild type strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27%) of the inulinase produced by the recombinant strain E136 was secreted into the culture. During the 10-1 fermentation,70.57±1.3 g/1 of pullulan in the fermented medium was attained from inulin (140.0 g/1) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase and most of the reducing sugars were used in pullulan production by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.It has been reported that the pullulan synthetase gene (PUL1) is involved in pullulan biosynthesis in yeasts. The ORF of the PUL1 gene in high pullulan producing yeast Aureobasidium melanogenum P16 strain was cloned and characterized. It was found that the ORF of PUL1 gene was 592 bp encoding 178 amino acid residues and was disrupted by an intron which had 55 bp. The promoter of the PUL1 gene had one CAAT box, one TATA box and one sequence 5’-HGATAR-3’. The deduced protein had a signal peptide of 18 amino acids and five putative N-glycosylation sites. After disruption of the PUL1 gene in strain P16, the recombinant DP108, one of the disruptants, produced 34.66±0.34 g/1 pullulan from sucrose, which is much less than that of its wild type strain P16, indicating that the PUL1 is related to the pullulan biosynthesis. The fused protein GFP-PUL1 was located on the surface of vacuoles in the yeast cells, suggesting pullulan biosynthesis happened in the cytosol of the yeast cells. After overexpression of the PUL1 gene, the tranformant G14 produced over 72.0 g/1 pullulan from sucrose while its wild type strain P16 only produced 65.53 g/1 pullulan under the same condition, indicating that pullulan production was greatly enhanced by the overexpression of the PUL1 gene encoding pullulan synthetase. Mw (the apparent molecular mass) of the purified pullulan was 4.422×105 (g/mol) and the pullulan had very narrow molecular mass distribution.