| The non-renewable nature of petroleum and pollution to environment during its transportation and usage have fostered research in development of revewable clean energy. Production of fuel ethanol as one of the renewable energy resources, especially fuel ethanol production using lignocellulosic feedstocks, has aroused special attention. Yeast strains of Saccharomyces cerevisiae are widely used for fuel ethanol production. However, cell viability and ethanol production efficiency of S. cerevisiae are repressed by various environmental stress conditions occurred during fuel ethanol production. These stressors include toxic level of acetic acid and other inhibitory compounds, high temperature, as well as high concentration ethanol. Therefore, it is of great significance to develop yeast strains with improved stress tolerance to enhance fuel ethanol production.In the previous studies in our lab, zinc sulfate supplementation in the culture broth improved environmental stress tolerance of the flocculating yeast SPSC01. However, the underlying mechanisms remain unclear. Subsequent comparative transcriptomic and proteomic analysis revealed the increased transcription levels of some key genes and proteins. Among these genes, CAR1, YHB1, ATX1 and PRB1 which are involved in different metabolic pathways were selected for further analysis in this thesis. CAR1 encodes arginase that hydrolyses arginine to ornithine; YHB1 encodes flavohemoglobin that has a relationship with NO metabolism; PRB1 encodes vacuolar proteases B, and ATX1 encodes a metal chaperonin related to copper transportation that effects the absorption of iron. Overexpression of these four genes was performed to explore the mechanisms underlying improved stress stress tolerance by zinc sulfate supplementation.Expression plasmids of the above-mentioned genes were constructed and transformed into an industrial S. cerevisiae strain Sc4126. At the same time, the empty plasmid with HO integration fragments was also transformed into the same strain to obtain a control strain. Subsequently, tolerance to environmental stresses of the recombinant strains was evaluated. It was found that comparing with the control strain, the recombinant yeast strains overexpressing CAR1, YHB1, PRB1 and ATX1 improved tolerance to acetic acid, ethanol, H2O2, and high temperature. In addition, ethanol fermentation performance of the recombinant strains was also improved in the presence of 5 g/L acetic acid, the fermentation time of the recombinant strains was shortened by at least 6 h. At high temperature of 41?, compared to the control strain, the recombinant strains can consume all the glucose in the medium, whereas high residual glucose was detected in the case of the control strain. Increased intracellular ornithine content was found in 4126-CAR1 strain under 5 g/L acetic acid, which is more significant than that under the control condition. Decreased amino acid content in the CAR1 overexpressing strain indicates that the remodeling of amino acid metabolism occurred under acetic acid stress. Metal content analysis indicated that iron content was increased and zinc content was reduced in 4126-ATX1 comparing with that of the empty vector control under acetic acid and thermal stress, indicating improved uptake of iron. The results in this study provide basis for further development of robust industrial yeast strains with improved stress tolerance and ethanol fermentation performance for more efficient ethanol using cellulosic feedstocks. |
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