Production of HM-1 killer toxin in Saccharomyces cerevisiae transformed with the PDR4 gene and δ-sequence-mediated multi-integration system

Abstract

The gene for the HM-1 killer toxin of Williopsis mrakii IFO 0895, which has a broad killer spectrum and is stable over wide pH and temperature ranges, was introduced into a killer-resistant mutant of Saccharomyces cerevisiae using a 2μm plasmid or δ-sequence-mediated multi-integration system under the control of the ADH1 promoter together with the PDR4 gene as a dominant selective marker. The killer activity of the transformants harboring the killer gene on a YEp plasmid was mitotically unstable and the plasmid copy number was reduced on a non-selective medium. In contrast, some transformants obtained using the δ-integration system showed strong killer activity which was mitotically stable even on a non-selective medium. Integration of multiple copies of the killer gene was confirmed by genomic Southern blot analysis. In a mixed culture experiment of the transformant using δ-mediated multi-integration which produces HM-1 and a HM-1-sensitive strain, Pichia anomala IFO 0569, the transformant prevented the growth of the latter.