BACKGROUND: Saccharomyces cerevisiae has been extensively employed as a host for the production of various biochemicals and recombinant proteins. The expression systems employed in S. cerevisiae typically rely on constitutive or galactose-regulated promoters, and the limited repertoire of gene expression regulations imposes constraints on the productivity of microbial cell factories based on budding yeast. RESULTS: In this study, we designed and characterized a series of allantoin-inducible expression systems based on the endogenous allantoin catabolic system (DAL-related genes) in S. cerevisiae. We first characterized the expression profile of a set of DAL promoters induced by allantoin, and further combined with the galactose-inducible (GAL) system to create a highly responsive genetic switch that efficiently amplifies the output signals. The resulting allantoin-GAL system could give a ON/OFF ratio of 68.6, with 6.8-fold higher signal output over that of direct P CONCLUSIONS: In summary, we characterized several allantoin-inducible DAL promoters from budding yeast and further developed a layered allantoin-GAL system that utilizes the DAL2 promoter to regulate the galactose regulon in budding yeast. The resulting allantoin-GAL system could give an impressive ON/OFF ratio that surpassed the traditional P