Overexpression of the pullulan synthetase gene enhanced pullulan production and its molecular weight by a mutant of Aureobasidium melanogenum P16

The pullulan synthetase gene (PUL1), involved in pullulan biosynthesis in Aureobasidium species, remains poorly understood. The open reading frame (ORF) of the PUL1 gene from the high pullulan-producing yeast Aureobasidium melanogenum P16 strain was cloned and characterized. The ORF of the PUL1 gene was determined to be 592 bp in length, encoding 178 amino acid residues. It was observed that an intron of 55 bp disrupted the gene. The promoter of the PUL1 gene contained a CAAT box, a TATA box, and a 5’-HGATAR-3′ sequence. The deduced protein possessed a signal peptide comprising 18 amino acids and harbored five potential N-glycosylation sites. Following the disruption of the PUL1 gene in strain P16, the disruptant DP108 yielded 34.7 ± 0.3 g/L of pullulan from sucrose, significantly lower than the production by its wild-type strain P16. This discrepancy underscored the close association between the PUL1 gene and pullulan biosynthesis. The majority of the fused Gfp-Pul1 proteins were found to be localized in the cell membrane and on the surface of vacuoles within the yeast-like fungal cells, indicating that pullulan biosynthesis occurred at these subcellular sites. Following the overexpression of the PUL1 gene, strain G14 produced >72.0 g/L of pullulan from sucrose, surpassing the production of its wild-type counterpart strain P16, which yielded 65.5 g/L of pullulan under the identical conditions. This outcome demonstrated that the overexpression of the PUL1 gene significantly enhanced pullulan production. The apparent molecular mass of the purified pullulan increased to 4.4 × 10⁵ Da. As an auxiliary protein, Pul1 was predicted to bind to AmAgs2, the key enzyme in pullulan biosynthesis, facilitating enhanced pullulan production.