Reprogramming of ethanol stress response in S. cerevisiae by the transcription factor Znf1 and the effect on the biosynthesis of glycerol and ethanol.

Samakkarn, Wiwan; Ratanakhanokchai, Khanok; Soontorngun, Nitnipa

High ethanol can severely inhibit the growth of yeast cells and fermentation productivity. The ethanologenic yeast Saccharomyces cerevisiae activates several well-defined cellular mechanisms of ethanol stress response (ESR); however, the involved regulatory control remains to be characterized. Here, we report a new transcription factor of ethanol stress adaptation called Znf1. It plays a central role in ESR by activating genes for glycerol and fatty acid production (GUP1, GPP1/2, GPD1, GAT1, and OLE1) to preserve plasma membrane integrity. Importantly, Znf1 also activates genes implicated in cell wall biosynthesis (FKS1, SED1, and SMI1) and the unfolded protein response (HSP30/104, KAR1, and LHS1) to protect cells from proteotoxic stress. The znf1Δ strain displays increased sensitivity to ethanol, ER-stressor β-mercaptoethanol, and cell wall-perturbing agent calcofluor white. To compensate for defective cell wall, the strain lacking ZNF1 or its target SMI1 displays increased glycerol levels of 16.4% and 29.2%, respectively. Znf1 collectively regulates an intricate network of target genes essential for growth, protein refolding, and production of key metabolites. Overexpression of ZNF1 not only confers tolerance to high ethanol but also increases ethanol production by 4.6% (8.43 g/L) or 2.8% (75.78 g/L) when using 2% or 20% (w/v) glucose as a substrate, respectively, compared to the wild-type strain. Mutually, stress-responsive transcription factors Msn2/4, Hsf1, and Yap1 are associated with some promoters of Znf1's target genes to promote ethanol stress tolerance. In conclusion, this work has implicated the novel regulator Znf1 in coordinating expression of ESR genes and illuminates the unifying transcriptional reprogramming during alcoholic fermentation. IMPORTANCE The yeast S. cerevisiae is a major microbe widely used in food and non-food industries. However, accumulation of ethanol has a negative effect on its growth and limits ethanol production. Znf1 transcription factor has been implicated in utilization of different carbon sources, including glucose, the most abundant sugar on earth, and non-fermentable substrates, as a key regulator of glycolysis and gluconeogenesis. Here, role of Znf1 in ethanol stress response is defined. Znf1 actively reprograms expression of genes linked to UPR, heat shock response, glycerol and carbohydrate metabolism, and biosyntheses of cell membrane and cell wall components. A complex interplay among transcription factors of ESR indicates transcriptional fine-tuning as the main mechanism of stress adaptation, and Znf1 plays a major regulatory role in the coordination. Understanding the adaptive ethanol stress mechanism is crucial to engineering robust yeast strains for enhanced stress tolerance or increased ethanol production.