Probing the limits of expression levels by varying promoter strength and plasmid copy number in Saccharomyces cerevisiae

Abstract

Heterologous gene expression levels were measured in yeast using the Escherichia coli gusA gene (encoding β- d-glucuronidase) as a reporter. The influence of two major parameters, promoter activity and plasmid copy number, was studied. (1) Promoters used in this study ranged from the very weak constitutive KEX2, the regulated CYC1 and PGK and the mating type-specific MFα1 to the strong constitutive TEF1 and TDH promoters. Using centromeric vectors, gusA expression levels varied within three orders of magnitude. (2) Plasmid copy number was changed by shifting from a monocopy (centromeric plasmid) over a moderate copy number (2μ-based plasmid) to a high copy number (2μ associated with the URA3-d selection marker), gusA expression levels increased relatively with plasmid copy number in all cases studied, but did not exceed the equivalent of 2% of total soluble yeast proteins. Coupling these variables, a 5-log range in gene expression levels was covered. Taken together, these results provide a framework which allows a comparison of existing and new promoters. This framework will be useful for expressing genes to required levels.