Expression of the Klebsiella pneumoniae pullulanase-encoding gene in Saccharomyces cerevisiae

Abstract

A 3800-base pair (bp) DNA fragment encoding the mature pullulanase from Klebsiella pneumoniae was inserted between two different yeast expression-secretion cassettes and an yeast gene terminator. These cassettes were cloned into an yeast centromeric plasmid YCplacIII and transformed into laboratory strains of Saccharomyces cerevisiae. Transcription initiation signals were derived from the mating pheromone alpha-factor (MF alpha 1p) and alcohol dehydrogenase (ADC1p) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). Transcription termination was effected by the yeast tryptophan synthase gene terminator (TRP5T). Southernblot analysis confirmed the presence of pulA in transformed yeasts and Northern-blot analysis revealed the presence of PUL1 mRNA. A pullulan agarose assay indicated the extracellular production of biologically active pullulanase by S. cerevisiae.