Breeding And Molecular Tolerance Mechanism Of Multiple Stress-tolerant Saccharomyces Cerevisiae Strains For Fuel Ethanol Production

With the growing energy shortage and environmental pollution,it is urgent to develop green and renewable energy.The production of fuel ethanol from amount of accumulated organic wastes(straws,molasses,etc.)can alleviate the crisis of energy and environment.If very high gravity(VHG)fermentation and high-temperature fermentation can be widely applied to industrial fuel ethanol production,the cost,wastewater discharge,and environmental pollution will be significantly reduced.Therefore,it is of great economic and environmental significance to breed industrial Saccharomyces cerevisiae(S.cerevisiae)strains with tolerance to multiple environmental stresses such as high ethanol,high temperature,high sugar,high salt.A flocculating industrial S.cerevisiae KF-7,which has good ethanol fermentation capacity and stress tolerance,was used as the original strain.Mutants with improved ethanol-or heat-tolerance were obtained by combining Atmospheric Room Temperature Plasma(ARTP)mutagenesis with several rounds of genome shuffling.Ethanol-and heat-tolerant mutants were then hybridized and the hybrid E-158 having multiple stress-tolerance was screened out.The molecular mechanisms of the multiple stress-tolerant strain E-158 under high ethanol,high temperature,high glucose,high salt,and other stresses were systematically analyzed by comparative transcriptomics.Based on the results of comparative transcriptomics,potential genes that may be related to the multiple stress tolerance of yeasts were identified.CRISPR/Cas9genome-editing technology was used to regulate the expression of the target genes.The effects of genes on the multiple tolerant phenotypes of strains were evaluated.The abilities of the constructed strains to use straws,molasses,and cassavas for VHG fermentation and high-temperature fermentation were further evaluated.The main research contents and conclusions are as follows:(1)Atmospheric and room temperature plasma(ARTP)mutagenesis combined with genome shuffling was successfully applied to obtain mutants with improved ethanol-or heat tolerance.The ethanol tolerance of the original strain KF-7 was significantly improved after ARTP mutagenesis,while the heat tolerance of KF-7 was slightly improved.Ethanol or heat tolerance of mutants was further enhanced after several rounds of genome shuffling.The best ethanol-tolerant strain C4-184 was screened out,and its ethanol production was 53.54% higher than that of KF-7 when batch fermentation was performed for 96 h using 141.83 g/L glucose with 9%(v/v)initial ethanol concentrations.The best heat-tolerant strain W3-9 was screened out,and its ethanol production was 15.59% higher than that of KF-7 when batch fermentation was performed for 72 h using 149.01 g/L glucose at 42℃.(2)Breeding of industrial S.cerevisiae with multiple stress-tolerance by hybridization.The best ethanol-and heat-tolerant mutants(C4-184 and W3-9)were hybridized,and a strain E-158 with multiple stress-tolerance was screened out.Strain E-158 grew better than the original strain KF-7 when exposed to multiple stress conditions.The concentrations of ethanol produced by enhanced strain E-158 during batch fermentation were 66.88%,33.36%,81.00%,10.14%,and 35.98% respectively higher than those produced by KF-7 under five stress conditions: 1)9%(v/v)initial ethanol,2)44°C,3)43°C and 3%(v/v)initial ethanol,4)27% YPD,5)1.25 M Na Cl.Furthermore,the level of Reactive Oxygen Species(ROS)of E-158 was significantly lower than that of KF-7 under each stress condition(e.g.,ethanol,heat,osmosis),while the Superoxide Dismutase(SOD)and Catalase(CAT)activities of E-158 were significantly higher than those of KF-7.One of the reasons why E-158 can resist multiple environmental stresses may be that it can rapidly scavenge more reactive oxygen species and has higher cellular antioxidant activity.The results of subcultivation showed that E-158 had good genetic stability.(3)Molecular mechanisms of multiple stress-tolerant strain E-158 under various stress conditions.There are some similarities as well as differences in the main metabolic pathways related to differentially expressed genes under different stress conditions.Under ethanol stress,the up-regulated genes were mainly involved in ion response,ion transport,transmembrane transport,and vitamin transport,while the down-regulated genes were mainly involved in DNA binding,asparagine metabolism,amino acid metabolism,and meiosis.Metabolic pathways related to oxidative phosphorylation,thermogenesis,cyanamide metabolism,and TCA cycle were significantly enriched.Under heat stress,the up-regulated genes were mainly involved in amino acid transport,transmembrane transport,and hexose transport,while the down-regulated genes were mainly involved in asparagine metabolism,amino acid metabolism,and ATPase activity.Pathways related to cyanamide metabolism,folate carbon pool,glyoxylate,and dicarboxylic acid metabolism,pyruvate metabolism were significantly enriched.Under the conditions of high temperature and ethanol stress,the up-regulated genes were mainly involved in the transport of copper ions,transmembrane transport,and oxidoreductase activity,while the down-regulated genes were mainly involved in the metabolism of nucleotides,amino acids,and purine nucleotides.Pathways related to alanine,aspartic acid and glutamic acid metabolism,folate carbon pool,cyanoamino acid metabolism were significantly enriched.Under high glucose stress,the up-regulated genes were mainly involved in iron balance,copper input,vitamin transport,and other metabolic processes,while the down-regulated genes were mainly involved in protein refolding,protein folding,and other metabolic processes.Pathways related to glycolysis/gluconeogenesis,endoplasmic reticulum protein processing,amino acid metabolism were significantly enriched.Under high salt stress,the up-regulated genes were mainly involved in sucrose decomposition,maltose metabolism,fatty acid metabolism,while the down-regulated genes were mainly involved in amino acid metabolism,asparagine catabolism.Pathways related to glycolysis/gluconeogenesis,methane metabolism,cyanoamino acid metabolism,pentose phosphate pathway were significantly enriched.In conclusion,the functions and metabolic pathways of differentially expressed genes were similar under ethanol,heat,and ethanol with heat stresses,while the functions and metabolic pathways of differentially expressed genes were similar under high glucose and high salt stresses.The common response pathways of strain E-158 related to central carbon metabolism,ROS response,oxidative phosphorylation,ribosome metabolism,amino acid metabolism,ion transport were enriched under all stresses.Also,according to the analysis of 28 common differentially expressed genes under all stresses,we speculated that transcription factors(TFs)Crz1p and Tos8 p,functional genes ASP3 and ENA5,unknown functional genes YOL162 W and YOR012 W may contribute to multiple tolerant phenotypes of yeasts.The analysis of the mentioned molecular mechanisms above provides potential target genes for the construction of multiple stress-tolerant strains by transcriptome-guided genetic engineering methods.(4)Effect of expression regulation of potential target genes on multiple tolerant phenotypes of strains.Using the original strain KF-7 as the host,the overexpression of Crz1 p or knockout of YOL162 W resulted in decreased tolerance to ethanol or high-temperature of KF-7,but the genetic modifications of these two genes can effectively improve the multiple tolerance of KF-7 under other stress conditions.Overexpression or knockout of other TFs or genes such as Tos8 p,ENA5,ASP3,YOR012 W was effective in enhancing the tolerance of KF-7 under multiple stresses.In conclusion,overexpression of gene ENA5 or transcription factor Crz1 p could significantly increase ethanol production and glucose consumption rate of KF-7 under various fermentation conditions.Moreover,overexpression of ENA5 in KF-7 had the best effect on improving the tolerance of it.Strain KEN5(overexpression of gene ENA5 in KF-7)has similar stress tolerance ability with multiple stress-tolerant strain E-158.Overexpression of transcription factor Crz1 p in E-158 could improve the multiple tolerance of it except for ethanol tolerance.Overexpression gene ENA5 in E-158 was effective in enhancing the tolerance of it under all stresses.Especially,when fermenting under the condition of 1.5 mol/L Na Cl,the ethanol concentration of stain(overexpression gene ENA5 in E-158)was about twice as high as that of E-158.(5)Different feedstocks were used to evaluate the VHG and hightemperature fermentation performance of the constructed strains.Using pretreated straws(solid content of 20%)as raw material,after simultaneous saccharification and fermentation at 42℃ for 96 h,the ethanol concentrations of strains KEN5,KAS11(knockout of ASP3 in KF-7),E-158,and EEN43 were 13.35%,17.09%,17.19%,22.38% respectively higher than that of KF-7.The ethanol concentration produced by strain EEN43 was 68.40 g/kg.After fermenting in 270.91 g/L molasses for 96 h,the ethanol concentrations of strains KEN5,E-158,and EEN43 were 22.32%,23.98%,27.31% respectively higher than that of KF-7.The ethanol concentration produced by strain EEN43 was 98.28 g/kg.Finally,when cassavas with a solid content of 35% was used for simultaneous saccharification and fermentation,the ethanol concentrations of strains KEN5(overexpression ENA5 in KF-7),E-158(multiple tolerant strain),and EEN43(overexpression ENA5 in E-158)were 6.50%,11.01%,14.08% respectively higher than that of KF-7.The ethanol concentration produced by strain EEN43 was 138.43 g/kg.In conclusion,in this study,a strain of S.cerevisiae strain E-158 with excellent multiple stress-tolerance was obtained through the combination of ARTP mutagenesis,genome shuffling,and hybridization.The molecular tolerance mechanisms of E-158 to various stresses were systematically revealed.The correlation of some potential target TFs,functional genes,and unknown functional genes with multiple tolerant phenotypes of S.cerevisiae was verified.The multiple tolerant industrial strains constructed in this study can be applied to the production of fuel ethanol from various raw materials including straws,molasses,and other organic wastes.The construction strategy of multiple stress-tolerant strains,the molecular tolerance mechanism of strains to various stresses,and target genes in this study will provide technical guidance,data basis,and theoretical basis for the construction of excellent strains for fuel ethanol production.