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

Bernard J H Janse*, Isak S. Pretorius

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

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 α-factor (MFα1p) and alcohol dehydrogenase (ADC1p) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone α-factor (MFα1s). 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.

Original languageEnglish
Pages (from-to)32-37
Number of pages6
JournalCurrent Genetics
Volume24
Issue number1-2
DOIs
Publication statusPublished - Jul 1993
Externally publishedYes

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