title: An improved system for estradiol-dependent regulation of gene expression in yeast.
abstract: BACKGROUND
  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. 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 beta-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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  The 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.
authors: Departamento de Genética, Universidad de Sevilla, Avda, Reina Mercedes 6, E41012-Seville, Spain. mjoseqc@us.es
authors: María J
authors: MJ
authors: Quintero
authors: Douglas
authors: D
authors: Maya
authors: Miguel
authors: M
authors: Arévalo-Rodríguez
authors: Angel
authors: A
authors: Cebolla
authors: Sebastián
authors: S
authors: Chávez