Abstract
BackgroundSaccharomyces cerevisiae is widely utilized in basic research as a model eukaryotic organism and in biotechnology as a host for heterologous protein production. Both activities demand the use of highly regulated systems, able to provide accurate control of gene expression in functional analysis, and timely recombinant protein synthesis during fermentative production. The tightly regulated GAL1-10 promoter is commonly used. However, induction of the GAL system requires the presence of the rather expensive inducer galactose and the absence of glucose in the culture media. An alternative to regulate transcription driven by GAL promoters, free of general metabolic changes, is the incorporation of the hybrid Gal4-ER-VP16 protein developed by D. Picard. This chimeric protein provides galactose-independent activation of transcription from GAL promoters in response to β-estradiol, even in the presence of glucose. However, constitutive expression of this transactivator results in relatively high basal activity of the GAL promoters, therefore limiting the gene expression capacity that is required for a number of applications.ResultsIn order to improve this expression tool, we have introduced additional regulatory elements allowing a simultaneous control of both the abundance and the intrinsic activity of the Gal4-ER-VP16 chimeric transactivator. The most efficient combination was obtained by placing the coding sequence of the hybrid activator under the control of the GAL1 promoter. This configuration results in an amplification feedback loop that is triggered by the hormone, and ultimately leads to the enhanced regulation of recombinant genes when these are also driven by a GAL1 promoter. The basal expression level of this system is as low as that of native GAL-driven genes in glucose-containing media.ConclusionThe feedback regulatory loop that we have engineered allows a 250-fold induction of the regulated gene, without increasing the basal activity of the target promoter, and achieving a 12-fold higher regulation efficiency than the previous configuration.
Highlights
Saccharomyces cerevisiae is widely utilized in basic research as a model eukaryotic organism and in biotechnology as a host for heterologous protein production
A native GAL promoter does not eliminate the basal increase produced by Gal4-estrogen receptor (ER)-VP16 In order to explore possible ways to improve the efficiency of the yeast estrogen-regulated system described by Louvion et al [17], we first tested if the high basal activity levels reported by these authors were due to the use of artificial GAL promoters, combining a number of Gal UAS fused to the CYC1 TATA sequences
A native GAL promoter would not improve the regulation capacity of an expression system based on the Gal4-ER-VP16 protein
Summary
Saccharomyces cerevisiae is widely utilized in basic research as a model eukaryotic organism and in biotechnology as a host for heterologous protein production Both activities demand the use of highly regulated systems, able to provide accurate control of gene expression in functional analysis, and timely recombinant protein synthesis during fermentative production. S. cerevisiae has been widely employed as a host organism in the expression of heterologous proteins [1,2,3,4,5,6,7], using regulated systems developed to allow low basal, highly inducible protein production In this sense, timely expression during fermentation is important to prevent a premature metabolic burden and any possible toxic effect throughout the culture growing-phase that might lead to a reduction in protein yields, or to genetic instability. Other yeast systems designed for tightly regulated gene expression incorporate transcriptional elements derived from bacteria, like those inducible or repressible by tetracycline (TetOff and Tet-On) [13,14]
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