Genomic Promoter Replacement Cassettes to Alter Gene Expression in the Yeast Saccharomyces cerevisiae

Promoter substitutions are frequently used to regulate the expression of genes in a specific manner such as for their conditional expression or for their overexpression. Chromosomal integration of a regulatable promoter upstream of an open reading frame (ORF) by homologous recombination using PCR-based gene targeting is straightforward and enables stable alterations of the genome. Furthermore, together with the promoter exchange, the target proteins can be tagged N-terminally with an epitope or a fluorescent protein. Expression levels can be constitutively lowered or increased by using promoters of different strengths. Reversible regulation of gene expression at the level of transcription can be achieved by using either regulatable yeast-endogenous promoters (e.g., GAL1-10 ) or heterogeneous promoters with synthetic transcription factors (e.g., TetO ). To regulate gene expression at the translational level, insertion of tetracycline-binding aptamers into the 5′ untranslated region (5′ UTR) of target genes can be used.