Construction And Fermentation Characteristics Of High Alcohol Tolerance Saccharomyces Cerevisiae

The development of fuel ethanol,which is environmental-friendly,could to some extent relieve the shortage of energy in our counrtry and the deterioration of environment conditions,as well as of in favor of food saftey.Saccharomyces cerevisiae is the most important strain in the fermentation for ethanol production,whereas high gravity ethanol or even very high gravity?VHG?fermentation could significantly improve the final titre of ethanol,with a sharp cut in the cost of energy consumption in the following step of separation.However,the stress caused either by the high concentration of substrate in the beginning or the product close to the end do harm to cell viability,leading to the death of cells under some circumstances,signifiantly negatively effecting the productivity.So it's of importance to uncover the mechanism of stress response to ethanol and construct a robust strain for ethanol production with enhanced stress tolerance.While the phenotype of ethanol tolerance in yeast is controlled by cross-talk of a bundle of genes and metabolic networks involved,thus conventional single gene knock-out or over-expression don't function in most cases here.In previous studies,it was found that cellular polarization would be altered for S.cerevisiae when undergoing a long-term exposure to ethanl,and furthermore,the cells with more significant alteration in shapes exhibited a higher survival rate,giving clues that Rho GTPase could serve as a potential regulation target for cellular ethanol response,just considering the central role of Rho GTPase as a key regulator of cell morphology.The family of Rho GTPase impose their biological effects at multi-levels involving many cellular signal pathways,characterized as a“global regulator”.Similar to global transcription machinary engineering?gTEM?,this research focused on the evolution of the key gene RHO1 by random mutation method,obseving the change in ethanol tolerance of the cells.Firstly,the Rho1 constitutive expression vector was constructed via the ligation of P_PGK1to the pYES2.0 vector backbone,then the library of Rho1 mutants was established by error-prone PCR followed by transformation into S.cerevisiae S288C.M5 was selected among the mutants with most significantly enhanced ethanol tolerance.The OD600 value of M5 was 1.38-folds of that of the control?with an empty pYES 2 vector?when cultured in10%?v/v?ethanol for 72 hours;And in the fermentation test with 300 g/L initial glucose,the final ethanol concentration of M5 was 21.7 g/L higher than the control;Besides,M5 also exhibited less sensitivity and higher tolerance to other stresses,such as acetic acid and oxidation stress?hydrogen peroxide?widely spread during fermentation process.At the same time,the strain overexpressing the native Rho1 didn't performe better than the control in both fermentation or under various stresses.Sequencing proved 3 mutation sites in Rho1 amino acids chain.The results of this paper showed that the evolution of Rho1 could improve both fermentation ability and ethanol tolerance of a yeast simultaneously,providing a new regulation target for enhancing yeast stress tolerance,and a novel perspective in microbial metabolic engineering.