New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

Reprogramming of the Ethanol Stress Response in Saccharomyces cerevisiae by the Transcription Factor Znf1 and Its Effect on the Biosynthesis of Glycerol and Ethanol.

High ethanol levels 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, GPP2, GPD1, GAT1, and OLE1) to preserve plasma membrane integrity. Importantly, Znf1 also activates genes implicated in cell wall biosynthesis (FKS1, SED1, and SMI1) and in the unfolded protein response (HSP30, HSP104, KAR1, and LHS1) to protect cells from proteotoxic stress. The znf1Δ strain displays increased sensitivity to ethanol, the endoplasmic reticulum (ER) stressor ß-mercaptoethanol, and the cell wall-perturbing agent calcofluor white. To compensate for a defective cell wall, the strain lacking ZNF1 or its target SMI1 displays increased glycerol levels of 19.6% and 27.7%, 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 levels but also increases ethanol production by 4.6% (8.43 g/liter) or 2.8% (75.78 g/liter) when 2% or 20% (wt/vol) glucose, respectively, is used as a substrate, compared to that of the wild-type strain. The 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 implicates 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 that is widely used in food and nonfood industries. However, accumulation of ethanol has a negative effect on its growth and limits ethanol production. The Znf1 transcription factor has been implicated as a key regulator of glycolysis and gluconeogenesis in the utilization of different carbon sources, including glucose, the most abundant sugar on earth, and nonfermentable substrates. Here, the role of Znf1 in ethanol stress response is defined. Znf1 actively reprograms expression of genes linked to the unfolded protein response (UPR), heat shock response, glycerol and carbohydrate metabolism, and biosynthesis 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.