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.

Increased production of isobutanol from xylose through metabolic engineering of Saccharomyces cerevisiae overexpressing transcription factor Znf1 and exogenous genes.

Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron-sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron-sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809 ± 0.400 g/L was achieved, following addition of 0.05 g/L FeSO4.7H2O in 5 L bioreactor. It corresponded to 155.88 mg/g xylose consumed and + 264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose.

Activation of cryptic xylose metabolism by a transcriptional activator Znf1 boosts up xylitol production in the engineered Saccharomyces cerevisiae lacking xylose suppressor BUD21 gene.

BACKGROUND: Xylitol is a valuable pentose sugar alcohol, used in the food and pharmaceutical industries. Biotechnological xylitol production is currently attractive due to possible conversion from abundant and low-cost industrial wastes or agricultural lignocellulosic biomass. In this study, the transcription factor Znf1 was characterised as being responsible for the activation of cryptic xylose metabolism in a poor xylose-assimilating S. cerevisiae for xylitol production. RESULTS: The results suggest that the expression of several xylose-utilising enzyme genes, encoding xylose reductases for the reduction of xylose to xylitol was derepressed by xylose. Their expression and those of a pentose phosphate shunt and related pathways required for xylose utilisation were strongly activated by the transcription factor Znf1. Using an engineered S. cerevisiae strain overexpressing ZNF1 in the absence of the xylose suppressor bud21Δ, xylitol production was maximally by approximately 1200% to 12.14 g/L of xylitol, corresponding to 0.23 g/g xylose consumed, during 10% (w/v) xylose fermentation. Proteomic analysis supported the role of Znf1 and Bud21 in modulating levels of proteins associated with carbon metabolism, xylose utilisation, ribosomal protein synthesis, and others. Increased tolerance to lignocellulosic inhibitors and improved cell dry weight were also observed in this engineered bud21∆ + pLJ529-ZNF1 strain. A similar xylitol yield was achieved using fungus-pretreated rice straw hydrolysate as an eco-friendly and low-cost substrate. CONCLUSIONS: Thus, we identified the key modulators of pentose sugar metabolism, namely the transcription factor Znf1 and the suppressor Bud21, for enhanced xylose utilisation, providing a potential application of a generally recognised as safe yeast in supporting the sugar industry and the sustainable lignocellulose-based bioeconomy.

Selection of Potential Yeast Probiotics and a Cell Factory for Xylitol or Acid Production from Honeybee Samples.

Excessive use of antibiotics has detrimental consequences, including antibiotic resistance and gut microbiome destruction. Probiotic-rich diets help to restore good microbes, keeping the body healthy and preventing the onset of chronic diseases. Honey contains not only prebiotic oligosaccharides but, like yogurt and fermented foods, is an innovative natural source for probiotic discovery. Here, a collection of three honeybee samples was screened for yeast strains, aiming to characterize their potential in vitro probiotic properties and the ability to produce valuable metabolites. Ninety-four isolates out of one-hundred and four were able to grow at temperatures of 30 °C and 37 °C, while twelve isolates could grow at 42 °C. Fifty-eight and four isolates displayed the ability to grow under stimulated gastrointestinal condition, at pH 2.0-2.5, 0.3% (w/v) bile salt, and 37 °C. Twenty-four isolates showed high autoaggregation of 80-100% and could utilize various sugars, including galactose and xylose. The cell count of these isolates (7-9 log cfu/mL) was recorded and stable during 6 months of storage. Genomic characterization based on the internal transcribed spacer region (ITS) also identified four isolates of Saccharomyces cerevisiae displayed good ability to produce antimicrobial acids. These results provided the basis for selecting four natural yeast isolates as starter cultures for potential probiotic application in functional foods and animal feed. Additionally, these S. cerevisiae isolates also produced high levels of acids from fermented sugarcane molasses, an abundant agricultural waste product from the sugar industry. Furthermore, one of ten identified isolates of Meyerozyma guilliermondiii displayed an excellent ability to produce a pentose sugar xylitol at a yield of 0.490 g/g of consumed xylose. Potentially, yeast isolates of honeybee samples may offer various biotechnological advantages as probiotics or metabolite producers of multiproduct-based lignocellulosic biorefinery.

Overexpression of Transcription Factor ZNF1 of Glycolysis Improves Bioethanol Productivity under High Glucose Concentration and Enhances Acetic Acid Tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae offers an attractive platform for synthesis of biofuels and biochemical; however, robust strains that can withstand high substrate concentration and fermentation conditions are required. To improve the yield and productivity of bioethanol, modification of glucose metabolism and cellular stress adaptation is investigated. Specifically, the role of Znf1 transcription factor in metabolic regulation of glucose is characterized. Here, Znf1 is first shown to activate key genes in glycolysis, pyruvate metabolism, and alcoholic fermentation when glucose is provided as the sole carbon source. Under conditions of high glucose (20 g L-1 ), overexpression of ZNF1 accelerated glucose consumption with only 0.67-0.80% of glucose remaining after 24 or 36 h of fermentation. Importantly, ZNF1 overexpression increases ethanol concentrations by 14-24% and achieves a maximum ethanol concentration of 76.12-88.60 g L-1 . Ethanol productivity is increased 3.17-3.69 in strains overexpressing ZNF1 compared to 2.42-3.35 and 2.94-3.50 for the znf1Δ and wild-type strains, respectively. Moreover, strains overexpressing ZNF1 also display enhanced tolerance to osmotic and weak-acid stresses, important trait in alcoholic fermentation. Overexpresssion of key transcriptional activators of genes in glycolysis and stress responses appears to be an effective strategy to improve bioethanol productivity and enhance strain robustness.