Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose

Abstract

The promoters of the highly expressed and stringently regulated GAL genes of Saccharomyces cerevisiae, are useful for expressing proteins in this organism. However, two problems complicate their use. First, because growth on glucose causes prolonged repression of GAL expression, cells are most rapidly induced after growth on nonfermentable carbon sources, conditions which usually support poor growth. Second, because the inducer of the GAL genes (galactose) also serves as a carbon source, the level of inducer is continually diminishing during growth of a Gal + strain, which may lead to reduced GAL expression. To solve the first problem, we have employed strains that carry the regl-501 mutation, which eliminates glucose repression of GAL expression. This gene has been shown to be located on the right arm of chromosome IV, distal but tightly linked to the TRP1 gene. We demonstrate that expression from GAL promoters is efficiently and rapidly induced in these reg1 strains by the addition of galactose to a culture growing in glucose medium. Levels of galactose as low as 0.02% can be used to obtain a 1500-fold induction of gene expression from GAL promoters in this strain. To surmount the second problem, we have used a gal1 mutant, deficient in the enzyme that catalyzes the first step of galactose utilization. We show that high levels of expression from GAL promoters are achieved rapidly in these mutants, for which galactose is a gratuitous inducer. With the goal of producing proteins during continuous fermentation of immobilized, nondividing cells, we also show that two proteins, intracellular bacterial β-galactosidase and secreted mouse amylase, are produced efficiently in reg1-501 cells arrested with the yeast peptide hormone, α-factor.