Mining Of Novel Functional Genes Involved In Acetic Acid Stress Tolerance Of Saccharomyces Cerevisiae And Construction Of Yeast Strains With Improved Performance

Stress tolerance of Saccharomyces cerevisiae is one of the important traits for its industrial applications.Superior stress tolerance of S.cerevisiae enables better fermentation efficiency under industrial conditions and benefits cell recycling.Therefore,studying mechanisms underlying stress tolerance is of importance to improve the production efficiency.Acetic acid is one of the important inhibitors generated from pretreatment of lignocellulosicbiomass.When present in high concentration,it severly represses yeast cell growth and fermentation.Therefore,improvement of acetic acid tolerance of S.cerevisiae benefits efficient cellulosic ethanol production.It was showed that zinc sulfate addition affected the function of membrane and membrane proteins.In this study,genes ADY2,ATO2 and JEN1 which encode carboxylic acid transporters significantly downregulated in S.cerevisiae cells with zinc sulfate addition.Meanwhile,content of intermediate metabolites were affected with zinc sulfate addition,such as increased content of 6-phophased glucose,alanine and glutathione.Transcription level of key genes involved in central carbon metabolism,amino acid metabolism and nucleotide metabolism,such as ADE1,ADE13 and ADE17 were significantly changed with zinc sulfate addition.In addition,relative expression level of genes(such as PPR1 and SET5)which encode zinc finger proteins was also affected.Hence,we supposed that the changes in membrane transporter,purine metabolisms,and regulatory proteins are critical for acetic acid resistance of yeast.Firstly,it was found that deletion of ADY2 improved growth under various stresses.The maximum specific growth rate was increased by ADY2 deletion in the presence of acetic acid,hydrogen peroxide and formic acid,which was 39.55%,23.41%and 15.86%,respectively,whereas no influence in the control condition without stress was observed by ADY2 deletion.Meanwhile,in the presence of 3.6 g/L acetic acid and mixed inhibitors,ethanol productivity of mutant strain BYady2 was higher than the control strain,which was 14.66%and 4.64%increase comparing to the control culture.In addition,the beneficial effect of ADY2 deletion on acetic acid tolerance was also indicated in industrial strain Sc4126.ADY2 deletion decreased intracellular acetic acid content,which was form 0.39 g/gDCW of control cells to 0.34 g/gDCW.At the same time,decreased membrane permeability was also observed by ADY2 deletion,suggesting that deletion of Ady2p improved stress tolerance by affecting membrane properties.Next,it was shown that overexpression of genes ADE1,ADE13 and ADE17 improved growth ability and fermentation efficiency under stress conditions.In the presence of 5 g/L acetic acid,mutants BADE 1,BADE 13 and BADE 17 consumed all glucose at 35 h,whereas still 3.95 g/L residual glucose existed in the control strain.Among the three strains,BADE 17 showed the best fermentation capability,which completed fermentation within 23 h.Meanwhile,in the simulated hydrolysate medium,the beneficial effect of ADE1,ADE13 and ADE17 overexpression was also indicated.Increased intracellular ATP content and activity of antioxidant enzyme SOD were observed in the three mutants,indicating that eliminating ROS by elevated expression of the ADE genes improved acetic acid stress tolerance.In addition,significantly increased GSH content was also observed in the ADE17 mutant,which contributed to further resistance to stress.On the other hand,overexpression of PPR1 and SET5 improved stress tolerance of acetic acid,H2O2 and high temperature.The recombinant strains BPPR1 and BSET5 also showed superior ethanol fermentation performance under acetic acid stress.Increased ethanol productivities of BPPR1 and BSET5 were achieved,which were 18.41%and 18.34%,respectively higher than that of the control strain BHO using corn stalk hydrolysate.Furhter studies showed that the ATP content and antioxidant enzyme activity were significantly increased by PPR1 and SET5 overexpression.In the meantime,transcription levels of stress responsive transcription regulators encoding genes HAA1,YAP1 and MSN4 were also elevated by overexpression of PPR1 and SET5,indicating that PPR1 and SET5 overexpression stimulated global stress response by modulating related transcription network.Set5p was identified as a methyltransferase of histone H4,but its zinc finger domains have not been studied.Therefore,key residues in the zinc finger domains were mutated,and used to explore their roles in Set5p function.The zinc-finger domain deletion led to 42.53%and 35.02%decreased growth rate under acetic acid and hydrogen peroxide stress conditions,suggesting that the zinc-finger domain exerted crucial role in Set5p to modulate stress tolerance.To further explore the underlying mechanisms of improved acetic acid tolerance by overexpression of SET5,comparative proteomic analysis was performed using iTRAQ.It was shown that 467 proteins were changed with Set5p overexpression in the presence of acetic acid,among which 380 proteins were upregulated and 87 downregulated.Functional category analysis revealed that proteins involved in glycolysis and PP pathway were upregulated,which was conducive to energy supply and precursor supplement.The upregulation of proteins involved in MAPK and TOR/ROS pathway represented a positive response to inhibitors in strain BSET5.In addition,SET5 overexpression promoted synthesis,processing and transport of proteins,which enable high efficiency and stability of cell growth and metabolism in the presence of acetic acid.The results in this thesis provide basis for further exploration of the molecular mechanisms underlying improved stress tolerance.Development of stress tolerant yeast strains based on the strategies in this work will further increase fuel ethanol production using cellulosic substrates.