| Acetic acid is one of the major inhibitory compounds for cell growth and ethanol fermentation of Saccharomyces cerevisiae, and is present in cellulosic hydrolysate as well as in industrial culture broth contaminated by acetic-acid producing bacteria. Therefore, improvement of acetic acid tolerance plays important role for efficient ethanol production. Although adjusting pH to5or6was proved to be effective to deal with acetic acid toxicity, this method is impractical for large scale industrial production due to the cost and increased risk of contamination. Zinc is an essential metal for yeast cell growth and metabolism, however, the important role of zinc is often neglected due to its status as a trace element. Previous studies in our group showed that zinc has protective effect on yeast ethanol tolerance and thermal tolerance of self-flocculating yeast SPSC01. In this study, the effect of different concentrations of zinc sulfate on ethanol production of self-flocculating yeast SPSC01in presence of high concentration of acetic acid was studied in batch fermentation in the3L bioreactor using100g/Lglucose. The results showed that zinc exhibits protective effect against acetic acid toxicity. Of the different concentrations of zinc sulfate tested,0.03g/L zinc sulfate showed the best protective effect. Addition of0.03g/L zinc sulfate shortened the lag phase time of cell growth of the self-flocculating yeast in presence of10g/L and15g/L acetic acid, and the fermentation time was also subsequently reduced. The length of lag phase was shortened nearly30h, with a27.83%increase in the biomass yield and a41.20%increase in the ethanol productivity comparing with the non-addition control. Analysis of by-products showed that biosynthesis of lactic acid was repressed by zinc addition, and significant reduction of propionic acid production was observed upon zinc addition, while no apparent effect was observed on succinic acid and citric acid biosynthesis. To further explore the molecular mechanisms of the zinc effect on the improvement of acetic acid tolerance, genome-wide transcriptional responses to0(control) and0.03g/L(expremental group) zinc sulfate were investigated in60,72and90h, respectively using RNA sequencing analysis under15g/L acetic acid. We found that385,2219and1253genes were differentilly expressed in response to0.03g/L zinc sultate compared to the non-addition control under acetic acid stress in60,72and90h respectively. Of the significantly changed genes, genes encoding ribosomal proteins, carbohydrate metabolism, amino acid metabolism and biosynthesis of cell wall were highly presented. Synthesis of ribosomal genes and ethanol fermentation related genes in the60and72h expression in early fermentation period indicated that the cell growth and fermentation condition were better in presence of zinc than that of the control group. Genes responsible for zinc transportation and storage were significantly upregulated in the control group comparing with the zinc-supplemented group, which indicates that yeast cells are under zinc deficiency condition in presence of high concentration of acetic acid. No remarkable changes of the genes encoding zinc finger proteins MSN2, STB5, MIG1, NRG1, UME6, RIM101which were reported to be related to acetic acid tolerance were observed, however, some of the downstream responsive genes of these zinc finger protein genes exhibited great differences in presence of zinc, which implied that the effect of zinc can be exerted on post-transcription or post translation level. Totally55significantly changed genes were first revealed in this study that response zinc supplementation, including several genes belonging to PAU family. The results presented in this study provide basis for further exploration of the molecular mechanisms underlying the protective effect of zinc on cell growth and ethanol fermentation in presence of acetic acid stress, and also shed light on the further metabolic engineering of industrial yeast for efficient fuel ethanol production. |
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