A new method for repeated "self-cloning" promoter replacement in Saccharomyces cerevisiae.

A method for repeated PCR-mediated promoter replacement in the yeast Saccharomyces cerevisiae is described. It was proposed to use the DNA fragment comprising the marker gene that enables both positive and negative selection (a selectable/counter-selectable marker) surrounded by direct repeats of the desired promoter as a promoter replacement cassette. This fragment is integrated upstream of the target gene because of PCR-added terminal sequences for homologous recombination with the target locus. Subsequent marker excision via homologous recombination between the copies of the two promoters leaves one copy of the desired promoter upstream of the target genes, without any heterologous scar sequence. To test this method, a set of plasmids bearing the S. cerevisiae URA3 gene surrounded by two copies of the ADH1 or PGK1 promoter was constructed. Using these cassettes, the native promoters of the GSH1 and GSH2 genes were replaced in the ura3Δ0 recipient strains. The proposed method is useful for research applications due to simple marker excision, and for construction of "self-cloning" industrial strains, because no heterologous DNA is retained in the genome of the resulting strain after marker excision.