Characterization of Aureobasidium Pullulans isolates based on multilocus sequence analyses and relationship between alpha amylase activities and pullulan profiles

Abstract

The fungus Aureobasidium pullulans is the major source of the commercial polysaccharide, pullulan, and also produces xylanase (EC 3.2.1.8). Tropical isolates of A. pullulans were isolated from various habitats. A total of 53 isolates used in this study included 45 new isolates from 15 provinces in Thailand, 1 new isolate from the U.S., and 7 comparative strains. These isolates were classified using multilocus sequence analyses from 5 loci including the rRNA ITS region, the rRNA IGS1 region, EF-1α, BT2, and RPB2. Based on the phylogenetic analyses, isolates were classified into 12 clades indicating that a vast diversity within the species. Moreover, morphological characteristics, pullulan production, and xylanase activity were determined for all isolates in an attempt to identify specific characteristics of each clade. The results showed different colors on different culture media, and some clades interestingly exhibited high levels of pullulan production and/or xylanase activity. Moreover, two new isolates were identified as a distinct species closely related to A. pullulans. To study the relationship between the activities of α-amylase and pullulan profiles, 5 representative strains were selected and grown in pullulan production medium. During cultivation, each culture broth was collected to assay the α-amylase and pullulanase activities and determine the pullulan profiles, including α-amylase sensitivity, pullulanase sensitivity, molecular weight, and viscosity. Pullulan yields gradually increased over time in individual strains while the molecular weight of pullulan decreased. However, the amylase activity of each strain was very low. It is possible that very low levels of amylase hydrolyze the minor maltotetraose structure of pullulan and cause the reduction of molecular weight, although it is also possible that other as yet uncharacterized enzymes or other factors play a role. Furthermore, the putative α-amylase gene of A. pullulans NRRL Y-12974 was characterized. The complete α-amylase gDNA contains 2,247-bp including 7 introns and 8 exons. The putative mRNA was 1,878-bp long encoding an α-amylase of 625 amino acid residues. Southern blot analysis indicated that there was only one copy of this gene in the genome. Finally, α-amylase mRNA was detected using reverse transcription-PCR, which indicated that there was α-amylase mRNA remaining in the cell during cultivation.