Synthetic Promoter Systems for Controlling Saccharomyces Cerevisiae Gene Expression
We characterized the previously reported artificial transcription factor Z3EV, which activates synthetic yeast promoters in response to the inducer beta-estradiol. We designed and constructed a series of synthetic promoters responsive to Z3EV and controlling expression of the reporter LacZ. We characterized their expression in different growth conditions, including aerobic glucose fermentation and growth on ethanol, and compared their expression levels to natural promoters from Saccharomyces cerevisiae.
We found that when Z3EV is expressed at moderate levels from the chromosome, the response to estradiol is very tight, and saturates at low levels of inducer. When Z3EV is expressed from a stronger natural promoter, its response to estradiol becomes more pronounced and the synthetic promoter expression increases with estradiol concentration. Higher expression is generally useful for genetic engineering applications, though for Z3EV this increased expression occurred at the cost of increased basal expression (leakiness) in the absence of estradiol inducer.
We examined the performance of the Z3EV system during the diauxic shift from glucose fermentation to growth on ethanol in aerobic batch culture. Many genes related to glycolysis are strongly expressed during glucose fermentation, but then decrease expression after glucose depletion and diauxic shift. We measured glucose consumption and ethanol production along with regulation by Z3EV of a LacZ reporter controlled by a synthetic promoter. We found that the Z3EV system increases gene expression after glucose has been depleted. This provides a tool for expression of heterologous genes at high levels after diauxic shift in batch culture.