Improving genetic tools to direct gene cloning in the osmoresistant yeast Zygosaccharomyces rouxii.
Lenka Pribylova (1), Jacky de Montigny (2), Serge Potier (2), Hana Sychrova (1)
(1) Dept. Membrane Transport, Inst. Physiology CzAcadSci, Videnska 1083, Prague, 14220, Czech Republic; (2) Lab. Microbiologie et Genetique, Inst. Botanique ULP-CNRS, 8 rue Goethe, 67083 Strasbourg, France
Z. rouxii, a species closely related to the model Saccharomyces cerevisiae, is widely known for its high osmotolerance. This particular feature is supposedly due to some sets of specific genes, which enable this yeast to grow in conditions restricted to most other yeast species (incl. S. cerevisiae ). Although a lot of attention was aimed to the physiological properties of Z. rouxii, the knowledge on the molecular level remains poor. Only a few genes have been characterized, and most of them were isolated by functional complementation of S. cerevisiae mutants. This situation is due mainly to the lack of tools for genetic engineering in Z. rouxii. So far, there existed only one type of Z. rouxii auxotrophic mutant ( leu2 ) and few suitable vectors. We developed an efficient and rapid protocol for transformation of Z. roxuii by electroporation and isolated first ura3 auxotrophic mutants. To improve tools for direct cloning in Z. rouxii, we have prepared a set of vectors for 1) gene expression - multicopy with replicon based on pSR1, monocopy (CEN S. cerevisiae /putative CEN Z. rouxii ), with different markers (auxotrophic - Sc URA3, Sc LEU2, Zr LEU2, dominant - kan MX, Sc MPR1 ) and promotors ( GAL1 ), and 2) gene deletion (loxP-kan MX -loxP cassette). This work was supported by grants GA CR 204/03/H066, 204/05/0028 and AVOZ 50110509 and French government (fellowship to L.P.).