| Acetic acid is one of the major by-products present in cellulosic hydrolysate, which is also generated during the ethanol fermentation of Saccharomyces cerevisiae. Cell viability, biomass accumulation and the utilization of fermentable sugars in the cellulosic hydrolysate are inhibited when high-concentration acetic acid is presented in the medium. Zinc finger proteins (ZFPs) play important roles in gene transcription and translation, and exert control on the regulation of multiple genes. Zinc finger motif and arrangement can be designed and engineered, and the artificial zinc finger protein can be used to regulate cell metabolism, which has beed widely used in basic research, drug design, and gene therapy. However, the in-depth studies on the application of zinc finger protein or similar artificial transcription factors (ATFs) in the metabolic engineering of industrial microorganisms are still limited.In this study, a novel ATFs expression library containing artificial zinc finger protein encoding genes was introduced the laboratory strain S288c, and one mutnt strain with higher acetic acid tolerance was selected, and the plasmid containing the zinc finger protein-encoding gene (ZFP-M01) was isolated, which was named pRS316-M01. Then the plasmid pRS316-M01 was transformed into an industrial yeast strain Sc4126, the yeast cells with pRS316-M01 were more tolerant to ethanol and acetic acid. The fermentation capacities were also compared. When the medium was supplemented with 5 g/L acetic acid, Sc4126-M01 showed a shorter lag phase and higher fermentation rate than Sc4126-M00. Interestingly, the acetic acid could also be metabolized at the end of fermentation for Sc4126-M01, and the secondary growth phenomenon was observed.Additionally, different treatment of acetic acid increased the catalase (CAT) activity and lowered reduced glutathione and glutathione disulfide (GSH/GSSG ratio), whereas decreased the superoxide dismutase (SOD) activity was observed in the zinc-finger protein-containing mutant, under the conditions of both with or without acetic acid treatments. It demonstrated that the strain containing ZFP-M01 could reduce more hydrogen peroxide, which resulted in less cell injury under acetic acid toxicity.The possible target genes containing the predicted binding sites of ZFP-M01 were identified from the genomic sequence of S. cerevisiae. Furthermore, three genes, namely. QDR3, IKSI and YFL040W, were found to have decreased transcription level in the presence of acetic acid in Sc4126-M01 comparing with those in the control strain. Deletion mutants of these three genes were further constructed, and it was found that QDR3 deletion mutant exhibited improved cell growth in the presence of acetic acid than that of the control strain, whereas deletion of IKS1 enabled higher resistance of the yeast cells upon acetic acid shock treatment. In addition, loss of YFL040W resulted in enhanced tolerance to oxidative stress which is related to acetic acid toxicity. The deletion mutants of QDR3, IKSl with the improved acetic acid tolerance were further tested in liquid fermentation experiments. Glucose consumption rate and ethanol productivity in the presence of acetic acid was improved in the QDR3 mutant compared to the wild type strain, whereas the IKS1 mutant showed retared growth. The results of this study demonstrate for the first time the involvement of QDR3, IKS1 and YFL040W in stress tolerance of S. cerevisiae, which provide basis for further the studies of functional genomics of S. cerevisiae. In addition, the studies in this thesis also add new insights on the regulation of transcription network by artificial transcription factor. |
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