Design and characterization of allantoin-inducible expression systems in budding yeast

Abstract

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_DAL2-controlled gene expression. Additionally, when a centromeric plasmid was used for EGFP expression, the ON/OFF ratio was increased to > 67.2, surpassing the EGFP expression levels driven by the DAL2 promoter. Subsequently, we successfully demonstrated that allantoin–GAL system can be used to effectively regulate carotenoid production and cell flocculation in S. cerevisiae.

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_DAL2-controlled gene expression. It is anticipated that utilizing our allantoin-inducible system in budding yeast with allantoin as the alternative nitrogen source might favor the low-cost production of biochemicals and pharmaceuticals.