Anna R. Oller

Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School



Aureobasidium pullulans, a polymorphic fungus, produces an exopolysaccharide called pullulan, which is composed of cross-linked maltotriose units. Pullulan is insoluble in ethanol and utilizes glucose, sucrose, or com syrup as it's carbon source. Pullulan can be used in many industrial and pharmaceutical applications, including an extended release tablet coating, a biodegradable plastic, a computer display binding agent, and an additive to low calorie foods. The genes and enzymatic pathways which control pullulan biosynthesis are currently unknown. This study was carried out to find a pullulan gene and create pullulan overproducers by using molecular biology techniques. RAPD-PCR using the 10-mer primer O-AB04, demonstrated different banding patterns among fungal strains. The bands obtained were excised from an agarose gel, purified, and transformed into the pullulan deficient strain B40203. Colonies which exhibited restored pullulan underwent further pullulan analysis. The amount of pullulan elaborated was determined by two methods. Filter weights of the ethanol precipitated pullulan were taken. Either neocuproine or pullulanase was used to quantify the amount of pullulan synthesized. A couple of mutants were found to have restored pullulan. The DNAs which restored pullulan synthesis were sequenced and compared to other known nucleotide sequences in Genbank. No homology was found to any other known nucleotide sequence. The DNA which restored pullulan synthesis was ligated, both forwards (sense) and backwards ( antisense ), into the yeast expression vector p YES2, which was cloned into E. coli cells. The plasmid DNA was then electroporated into competent Saccharomyces cerevisiae cells, and pullulan could be seen visually from the forward transformants. Gene expression was induced by media containing galactose and cells were taken at 0, 2, 4, 6, 8, and 10 hours after exposure to the galactose. RNA was then isolated and cDNA was made. The cDNA was then used in RAPD-PCR to determine the level of gene expression. A genomic D A library of A. pullulans will be screened with the gene to determine if more pullulan genes exist.

Library of Congress Subject Headings

Polysaccharides -- Synthesis
Pleomorphic fungi
Genetic transformation



Number of Pages



South Dakota State University