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

Abstract

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.

Keywords: Bioconversion; Metabolic engineering; Transcription factor Znf1; Xylitol; Xylose utilisation; Yeast.

Fig. 1

Expression levels of Znf1 target genes during glucose–xylose shift. Relative expression levels of genes involved in xylose metabolism, pentose phosphate pathway, glycolysis, gluconeogenesis, glycerol metabolism and TCA cycle were examined using RT-qPCR. Changes in the levels of mRNAs were indicated as following: 2% xylose shift in the wild-type strain (the X rectangle box), in the wild-type strain relative to the znf1∆ strain during the 2% xylose shift (the Y rectangle box) and, in the wild-type strain relative to the znf1∆ strain during growth in 2% glucose (the Z rectangle box). Green, red or yellow color box indicated genes whose expression was either activated/induced, repressed or unaltered by xylose or the transcription factor Znf1, respectively. The relative expression levels were obtained via the comparative C_t method for quantification of the ∆∆C_t values. Altered expression more than 2-folds was considered significant. The arrowheads in the figure represent the direction of enzymatic reactions. Abbreviations: ADH1, alcohol dehydrogenase I; ADH2, alcohol dehydrogenase II; BUD21, component of small ribosomal subunit/ xylose suppressor; COX1, cytochrome c oxidase; CYC3, cytochrome c heme lyase; ENO1, enolase; FBA1, fructose 1,6-bisphosphate aldolase; GCY1, glycerol dehydrogenase; GDH2, glutamate dehydrogenase; GLK1, glucokinase; GND1, 6-phosphogluconate dehydrogenase; GPD1, glycerol-3-P dehydrogenase; GPD2, glycerol-3-P dehydrogenase; GPM1, phosphoglycerate mutase; GPP1, glycerol-3-P phosphatase; GPP2, glycerol-3-P phosphatase; GRE3, aldose reductase; HXK1, hexokinase isoenzyme 1; HXK2, hexokinase isoenzyme II; PFK1, phosphofructokinase I; PFK2, phosphofructokinase II; PGI1, phosphoglucose isomerase; PGK1, 3-phosphoglycerate kinase; PDC1, pyruvate decarboxylase; PYC1, pyruvate carboxylase; PYK1, pyruvate kinase; RKI1, ribose-5-phosphate ketol-isomerase; RPE1, ribulose-5-phosphate 3-epimerase; SDH1, succinate dehydrogenase; SOL3, 6-phosphogluconolactonase; SOL4, 6-phosphogluconolactonase; SOR1, sorbitol dehydrogenase; SOR2, sorbitol dehydrogenase; TAL1, transaldolase; TDH1, glyceraldehyde-3-phosphate dehydrogenase; TKL1, transketolase; TKL2, transketolase; TPI1, triose phosphate isomerase; XKS1, xylulokinase; XYL2, xylitol dehydrogenase; YDL124W, NADPH-dependent alpha-keto amide reductase; YJR096W, xylose and arabinose reductase; YPR1, NADPH-dependent aldo–keto reductase; ZWF1, glucose-6-phosphate dehydrogenase. Compounds, AKG, α-ketoglutarate; 13BPG, 1,3-bisphosphoglycerate; CIT, citrate; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; FDP, fructose-1,6-diphosphate; F6G, fructose-6-phosphate; FUM, fumarate; GAP, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; G3P, glyceraldehyde-3-phosphate; ICI, isocitrate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; 6PGC, 6-phosphogluconate; 6PGL, 6-phospho-gluconolactonase; MAL, malate; OXA, oxaloacetate; R5P, ribose 5-phosphate; RL5P, ribulose 5-phosphate; S7P; sedoheptulose 7-phosphate; SUCCoA, succinyl CoA; SUCC, succinate; X5P, xylulose 5-phosphate

Fig. 2

Expression levels of genes involved in xylose metabolism in the ZNF1 overexpressing strain (ZNF1-OE) during the growth in 2% xylose and 0.05% glucose mix. A Relative expression levels of xylose metabolic genes (GCY1, GRE3, YDL124W, YPR1, XYL2, SOR1, SOR2 and BUD21 genes) B glycolytic and alcoholic fermentative genes (HXK1, HXK2, GPM1, ENO1, PYK1, PDC1, and ADH1 genes) C hexose transporter and PPP genes (HXT4, HXT7, GAL2, ZWF1, GND1, and TAL1 genes) in ZNF1-OE strain compared to the wild type strain during the growth in 2% xylose and 0.05% glucose mix. The relative expression levels were obtained via the comparative C_t method for quantification of the ∆∆C_t values. Altered expression levels more than 2-folds were considered significant. The average values were calculated from at least two independent experiments performed in three replicates. D Metabolic engineering strategy via overexpression of ZNF1 transcription factor gene to activate its target genes linked to xylose metabolism and deletion of xylose suppressor BUD21 in S. cerevisiae. The green arrow indicated induction of genes expressed by fold-changes which is compared to the wild-type BY4742 strain

Fig. 2

Expression levels of genes involved in xylose metabolism in the ZNF1 overexpressing strain (ZNF1-OE) during the growth in 2% xylose and 0.05% glucose mix. A Relative expression levels of xylose metabolic genes (GCY1, GRE3, YDL124W, YPR1, XYL2, SOR1, SOR2 and BUD21 genes) B glycolytic and alcoholic fermentative genes (HXK1, HXK2, GPM1, ENO1, PYK1, PDC1, and ADH1 genes) C hexose transporter and PPP genes (HXT4, HXT7, GAL2, ZWF1, GND1, and TAL1 genes) in ZNF1-OE strain compared to the wild type strain during the growth in 2% xylose and 0.05% glucose mix. The relative expression levels were obtained via the comparative C_t method for quantification of the ∆∆C_t values. Altered expression levels more than 2-folds were considered significant. The average values were calculated from at least two independent experiments performed in three replicates. D Metabolic engineering strategy via overexpression of ZNF1 transcription factor gene to activate its target genes linked to xylose metabolism and deletion of xylose suppressor BUD21 in S. cerevisiae. The green arrow indicated induction of genes expressed by fold-changes which is compared to the wild-type BY4742 strain

Fig. 3

Effect of low glucose–xylose mix on induction of xylose utilization in different S. cerevisiae strains. The ZNF1-OE strain overexpressing ZNF1 gene was investigated during culture in YPX containing 2% xylose (w/v) and low glucose at concentration of 0.05%, 0.04%, 0.03%, 0.02%, 0.01% (w/v) or without glucose expressed in term of cell dry weight (g/L) (A), Cell survival was analyzed using CFU/ml method (B), Glucose concentration (g/L) (C), Xylose concentration (g/L) (D), Phenotypic analysis on increased concentration of xylose (E). The wild-type BY4742, the znf1∆, the bud21∆, the rescued strain (znf1∆ + pLJ529-ZNF1) the overexpression ZNF1 (BY4742 + pLJ529-ZNF1 and ZNF1-OE), and the engineered bud21∆ + pLJ529-ZNF1 of S. cerevisiae strains were observed on YPX agar plates contained 0.05% glucose mixed with different concentration of xylose at 2 or 10% (w/v). Ten-fold serial dilutions of cells were spotted on plates and incubated at 30 °C for 2–5 days. Error bars indicated standard deviations calculated from at least two independent experiments performed in triplicate. Significance differences were determined by one-way ANOVA with Tukey HSD method (*, p < 0.05; **, p < 0.01)

Fig. 4

Xylose fermentation profile and xylitol production of S. cerevisiae wild type and engineered strains. Different S. cerevisiae strains of BY4742 + pRS316, BY4742 + pLJ529-ZNF1, bud21∆ + pRS316, and bud21∆ + pLJ529-ZNF1 were grown under YPX supplemented with A 2% (w/v) or B 10% (w/v) of xylose mixed with 0.05% glucose at 30 °C for 10 or 18 days, respectively. For all plots presented, xylose concentration (g/L) (solid line), xylitol concentration (g/L) (dashed line). Xylose consumption and xylitol concentrations were determined by HPLC, and data was based on two independent experiments conducted in triplicate. Error bars indicated standard deviations calculated from at least two independent experiments performed in triplicate. Significance differences were determined by one-way ANOVA with Tukey HSD method (*, p < 0.05; **, p < 0.01, as compared to the control BY4742 + pRS316 strain or ¥, p < 0.05 as compared to the control bud21∆ + pRS316)

Fig. 5

Overexpression of ZNF1 and deletion of BUD21 genes conferred tolerance to furfural and lignocellulosic acids stress. The S. cerevisiae wild-type BY4742, the znf1∆, the BY4742 + pRS316, the BY4742 + pLJ529-ZNF1, the bud21∆ + pRS316 and bud21∆ + pLJ529-ZNF1 strains were examined for growth and cell survival. A Growth assays were conducted. Cells were grown in YPX10 media containing 10% xylose (w/v) and 0.05% glucose plus 20 mM furfural (FF), 40 mM formic acid (FA), or 85 mM levulinic acid (LA). Growth of strains were monitored and expressed as the optical density values (OD₆₀₀) for 5 days at 30 °C. B Spot tests of different S. cerevisiae strains were examined on YPX10 plates containing10% xylose (w/v) and 0.05% glucose to monitor cell survival in the presence of 35 mM formic acid, 20 mM furfural, or 85 mM levulinic acid. Ten-fold serial dilutions of cells were spotted on plates and incubated at 30 °C for 2–3 days. C Cell survival was analyzed using CFU/ml method. Significance differences were determined by one-way ANOVA with Tukey HSD method (*, p < 0.05; **, p < 0.01) as compared to the controls BY4742 or BY4742 + pRS316. Error bars indicated standard deviation (SD)

Fig. 5

Overexpression of ZNF1 and deletion of BUD21 genes conferred tolerance to furfural and lignocellulosic acids stress. The S. cerevisiae wild-type BY4742, the znf1∆, the BY4742 + pRS316, the BY4742 + pLJ529-ZNF1, the bud21∆ + pRS316 and bud21∆ + pLJ529-ZNF1 strains were examined for growth and cell survival. A Growth assays were conducted. Cells were grown in YPX10 media containing 10% xylose (w/v) and 0.05% glucose plus 20 mM furfural (FF), 40 mM formic acid (FA), or 85 mM levulinic acid (LA). Growth of strains were monitored and expressed as the optical density values (OD₆₀₀) for 5 days at 30 °C. B Spot tests of different S. cerevisiae strains were examined on YPX10 plates containing10% xylose (w/v) and 0.05% glucose to monitor cell survival in the presence of 35 mM formic acid, 20 mM furfural, or 85 mM levulinic acid. Ten-fold serial dilutions of cells were spotted on plates and incubated at 30 °C for 2–3 days. C Cell survival was analyzed using CFU/ml method. Significance differences were determined by one-way ANOVA with Tukey HSD method (*, p < 0.05; **, p < 0.01) as compared to the controls BY4742 or BY4742 + pRS316. Error bars indicated standard deviation (SD)

Fig. 6

Solid state fermentation of rice straw pretreated with Xylaria sp. BCC1067 for xylose to xylitol conversion. A Enzyme activity of cellulase and xylanase (U/g) and sugar concentration (mg/g of rice straw) under solid-state fermentation. Rice straw was pretreated with fungi Xylaria sp. BCC1067 cultivated for 28 days at 25 °C at 70% moisture content for 28 days. The reaction mixture was incubated at 50 °C for 10 min. The reducing sugars released were quantified using glucose or xylose as a standard. B Conversion of xylose to xylitol from rice straw hydrolysate. The BY4742 + pRS316, the BY4742 + pLJ529-ZNF1, the bud21∆ + pRS316, and the bud21∆ + pLJ529-ZNF1 S. cerevisiae strains were grown using rice straw hydrolysate supplemented with YP medium and 0.05% glucose. Strains were incubated at 30 °C with shaking for 60 h. Glucose, xylose and xylitol concentrations were determined by HPLC and CDW (mg/L) was also obtained. Error bars indicated standard deviations calculated from at least two independent experiments performed in triplicate. Significance differences were determined by one-way ANOVA with Tukey HSD method (*, p < 0.05; **, p < 0.01) or (¥, p < 0.05) as compared to BY4742 + pRS316 or bud21∆ + pRS316, respectively