PRODUCTION OF HM-1 KILLER TOXIN IN SACCHAROMYCES-CEREVISIAE TRANSFORMED WITH THE PDR4 GENE AND DELTA-SEQUENCE-MEDIATED MULTI-INTEGRATION SYSTEM

The gene for the HM-1 killer toxin of Williopsis mrakii IFO 0895, whic h has a broad killer spectrum and is stable over wide pH and temperatu re ranges, was introduced into a killer-resistant mutant of Saccharomy ces cerevisiae using a 2 mu m plasmid or delta-sequence-mediated multi -integration system under the control of the ADH1 promoter together wi th the PDR4 gene as a dominant selective marker. The killer activity o f the transformants harboring the killer gene on a YEp plasmid was mit otically unstable and the plasmid copy number was reduced on a non-sel ective medium. In contrast, some transformants obtained using the delt a-integration system showed strong killer activity which was mitotical ly stable even on a non-selective medium. Integration of multiple copi es of the killer gene was confirmed by genomic Southern blot analysis. In a mixed culture experiment of the transformant using delta-mediate d multi-integration which produces HM-1 and a HM-1-sensitive strain, P ichia anomala IFO 0569, the transformant prevented the growth of the l atter.