Multicopy plasmids containing the gene for the transcriptional activator LAC9 are not tolerated by K. lactis cells.

The high copy number 2-microns DNA-like Kluyveromyces plasmid pKD1 was extremely unstable in Kluyveromyces lactis when carrying the gene for the regulatory protein LAC9, a transcriptional activator involved in the induction of the LAC and GAL genes. Transformants of a lac9 mutant strain normally contained rearranged plasmids and all were Lac-, indicating that the LAC9 gene was inactive. Lac+ "revertants" could be obtained from Lac- transformants by selection on lactose plates. In some of these, the pKD1-based plasmid was stably maintained by being integrated into the chromosome of the cell; in others, the disrupted chromosomal gene was restored by a gene conversion event. None of the Lac+ revertants had more than one intact LAC9 gene, an indication that LAC9 overexpression affects cell viability.