Transformation and Expression of a Pullulan Gene in Saccharomyces Cerevisiae. (Biology Section).

Oller, A.R. Department of Biology, Central Missouri State University. TRANSFORMATION AND EXPRESSION OF A PULLULAN GENE IN SACCHAROMYCES CEREVISIAE. Pullulan is a polysaccharide of great economic importance in pharmaceutical and industrial products. Previous studies identified a pullulan gene through Random Amplified Polymorphic DNAs (RAPD) Polymerase Chain Reaction (PCR) techniques, and the gene was transformed into pullulan deficient mutants created by gamma irradiation. Once restored pullulan synthesis was determined, the gene was sequenced. The present study was to determine whether ligation of the gene into the pYES2 yeast expression vector caused pullulan elaboration in S. cerevisiae, which is much easier to culture than the native fungus, Aureobasidium pullulans. The pullulan DNA was ligated both forward (sense) and backward (antisense) into the pYES2 through the use of several restriction endonucleases. The plasmid vector was then cloned into competent Escherichia coli cells. The presence of the desire d DNA was confirmed through plasmid isolation and use of restriction endonucleases. The plasmid DNA was then electroporated into competent Saccharomyces cerevisiae cells. The Saccharomyces transformants were then plated on Synthetic Media (SM) and Ueda media. Pullulan synthesis was visualized on Ueda plates. Transformants were visualized microscopically to confirm pullulan elaboration from the cell. Further, pullulan elaboration was determined by alcohol precipitation and filter weights. Reverse Transcriptase (RT)-PCR was performed on the transformants to confirm gene expression. The organisms containing the sense DNA were found to produce pullulan visually and microscopically, whereas the organisms containing the antisense DNA did not. The S. cerevisiae transformants were found to produce even greater quantities of the polysaccharide than Aureobasidium pullulans in the wild. RT-PCR further indicated gene expression of the sense DNA, but not the anti-sense DNA.