Improving Thermotolerance Of Saccharomyces Cerevisiae Based On Heat-induced Antioxidant Defenses

Energy crisis and environmental crisis force us to seek a new energy to replace the traditional fossil fuels. Non-grain fuel bioethanol, which achieve the harmonious development of energy, grain and environment, has been the focus of an alternative to traditional fossil fuels. However, high production costs restrict the development of nongrain fuel bioethanol industry. High-temperature fermentation can effectively reduce the cooling costs, equipment costs, ethanol extraction costs, and other potential costs in non-grain fuel bioethanol production process, and is the key to push forward non-grain fuel bioethanol industrialization. But, high temperature make yeast cells facing with severe oxidative stress, which lead to cell death. Based on this, the antioxidant proteins with different functions from Thermus thermophilus or Saccharomyces. cerevisiae were heterogenous expressed or overexpressed in industrial S. cerevisiae to improve its thermotolerance. The main results are as follows:(1) Antioxidant proteins with different functions were excavated from Thermus thermophilus or S. cerevisiae, based on the antioxidant defense system of S. cerevisiae, and were assembled with strong constitutive promoter FBA1 p to construction devices. Those divices were then implanted in recombinant plasmid form into industrial S.cerevisiae and obtaind 15 engineered S. cerevisiae, among of which the engeered S. cerevisiae S.c-TTHA0557 and S.c-SODA were constructed with plasmid from the previous work of our project group. All the 15 engineered S. cerevisiae include 4 containing superoxyde dismutase(SOD) devices, 1 containing peroxidase(Prx) device, 1 containing catalase(CAT) device, 4 containing thioredoxin(Trx) devices, 4 containing thioredoxin reductase(Trx R) devices, and 1 containing NAD(H) kinase device.(2) From the growth conditions and fementation characteristics level, the thermotolerance of the 15 engineered S. cerevisiae were characterized and discussed according to the results of growth curve, viable count, drip plate assay and ethanol production at 37 °C and 40 °C. Overexpression of SOD encoding genes sod1 or sod2 can significantly improve the growth conditions and ethanol production of Saccharomyces. cerevisiae. Compared to the control, the OD660 of S.c-SOD1 and S.cSOD2 were increased by 80.7% and 74% after 72 h respectively, the viable count were increased by 50.5% and 78% after 36 h respectively, and the ethanol production were increased by 826.1% and 74% after 12 h respectively, at 40 °C; The ethanol production and the thermotolerance to 50 °C of the engineered S. cerevisiae S.c-TTHA1300 with Prx devices were significantly improved. Compared to the control, the ethanol production of S.c-TTHA1300 was increased by 16% after 24 h, and the relative survival rate was increased by 289% after treated at 50 °C for 45 min. However, ttha1300 shows poor adaptation of S. cerevisiae and the growth of S.c-TTHA1300 was somewhat inhibited; Heterologous expression of Trx encoding genes ttha1747 or ttha1422 can significantly improve the growth conditions and ethanol production of S. cerevisiae. Compared to the control, the OD660 of S.c-TTHA1747 and S.c-TTHA1422 were increased by 51.5% and 58.5% after 72 h respectively, the viable count were increased by 89% and 93.6% after 36 h respectively, and the ethanol production were increased by 952.2% and 921.7% after 12 h respectively, at 40 °C; Moreover, compared to the control, the viable count of the engineered S. cerevisiae S.c-TTHA1215 and S.cTTHA1920 with Trx R devices was increased by 65.7% and 74.6% after 36 h respectively, and the ethanol production of. S.c-TTHA1215 and S.c-TTHA1920 was increased by 73.9 and 65.2% after 12 h respectively, at 40 °C. The viable count after 36 h and the highest ethanol production of the engineered S. cerevisiae S.c-Pos5 p with NAD(H) kinase devices were increased by 262.5% and 7.42% than control, but the ethanol synthesis rate of S.c-Pos5 p was significantly decreased at 40 °C. In conclusion, the thermotolerance and ethanol production of the engineered strain S.c-SOD1, S.cSOD2, S.c-TTHA1300, S.c-TTHA1747, S.c-TTHA1422, S.c-TTHA1215, S.cTTHA1920 and S.c-Pos5 p were improved in different degrees.