PROBING THE LIMITS OF EXPRESSION LEVELS BY VARYING PROMOTER STRENGTH AND PLASMID COPY NUMBER IN SACCHAROMYCES-CEREVISIAE

Heterologous gene expression levels were measured in yeast using the E scherichia coli gusA gene (encoding B-D-glucuronidase) as a reporter. The influence of two major parameters, promoter activity and plasmid c opy number, was studied. (1) Promoters used in this study ranged from the very weak constitutive KEX2, the regulated CYC1 and PGK and the ma ting type-specific MF alpha 1 to the strong constitutive TEF1 and TDH promoters. Using centromeric vectors, gusA expression levels varied wi thin three orders of magnitude. (2) Plasmid copy number was changed by shifting from a monocopy (centromeric plasmid) over a moderate copy n umber (2 mu-based plasmid) to a high copy number (2 mu associated with the URA3-d selection marker). gusA expression levels increased relati vely 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 expre ssing genes to required levels.