Abstract
BackgroundSynthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial applications. The human estrogen receptor α contains a ligand binding domain that enforces dimerization and nuclear import upon binding of the inducer 17β-estradiol. In this study, we tested the potential of this ligand binding domain to be used in filamentous fungi as an auto-regulatory domain in a synthetic transcription factor.ResultsWe constructed the synthetic transcription factor SynX by fusing the DNA-binding domain of Xyr1 (Xylanase Regulator 1), the transactivation domain of Ypr1 (Yellow Pigment Regulator 1), and the ligand binding domain of the human estrogen receptor α. SynX is able to strongly induce the gene expression of xylanases and an aldose reductase by addition of 17β-estradiol, but SynX does not induce gene expression of cellulases. Importantly, the induction of xylanase activities is mostly carbon source independent and can be fine-tuned by controlling the concentration of 17β-estradiol.ConclusionThe ability of SynX to induce gene expression of xylanase encoding genes by addition of 17β-estradiol demonstrates that the ligand binding domain of the human estrogen receptor α works in filamentous fungi, and that it can be combined with a transactivation domain other than the commonly used transactivation domain of herpes simplex virion protein VP16.
Highlights
Synthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial appli‐ cations
The tightness of the expression system and the ability of the SynX to induce expression of the main Xyr1 target genes were determined on transcript and enzymatic levels; further we studied the influence of different carbon sources and different concentrations of estradiol on the xylanolytic and cellulolytic activities
We decided to use 300 nM as standard working concentration because on xylan and lactose the measured xylanolytic activities were very close to the respective maximum activity (Fig. 5b), while on glucose and glycerol the maximum activities were reached at 17β-estradiol concentrations that we considered to be too high for a feasible application
Summary
Synthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial appli‐ cations. The human estrogen receptor α contains a ligand binding domain that enforces dimerization and nuclear import upon binding of the inducer 17β-estradiol. The expression should be tunable and reach a high expression level Such a system enables the expression of genes of interest at a desired time point to desired intensities, For filamentous fungi several such synthetic expression systems were developed previously. Upon binding of an inducer to the LBD, hERα undergoes a conformational change, which results in dimer stabilization, nuclear import, and exposure of surfaces for interactions with the basic transcription machinery (the LBD is responsible for transactivation) [6, 7, 10, 13, 14]. Refer to the review by Yaşar et al [15] for a summary about the current knowledge on the hERα
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