Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in...

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ-based plasmids with a choice of three inducible promoters, P(GAL1) , P(CUP1) and P(ADH2) . These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA-mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid-based and integrated chromosomal cassettes, and compared to that of the glycolytic P(PGK1) promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P(PGK1) , P(TEF1) and P(HXT7-391) .