| Isobutanol is regarded as a new clean biological energy and has been used in many fields.Saccharomyces cerevisiae（S.cerevisiae）can produce isobutanol through valine biosynthesis pathway.It is an effective way to increase the yield of isobutanol by over-expressing or deleting genes involved in valine biosynthesis pathway through genetic engineering.Isobutanol tolerances of engineered strains restricted further increasing isobutanol yield in S.cerevisiae.The goal of this work is to screen S.cerevisiae mutants with higher isobutanol tolerance by mutagenesis and increase isobutanol yield by genetic engineering technologies.Firstly,Chemical mutagen ethyl methane sulfonate（EMS）was used to induce the wild type W303-1A and select mutants.The initial EMS concentration was 40μL/mL.Five concentrations(OD60000 were 1,10-1,10-2,10-3,10-4)of mutants were set up as well.We screened 25 mutants that can grow in YPD medium containing 2%isobutanol and 7 mutants of them could grow in YPD medium containing 3%isobutanol.Finally,No.39 mutant（EMS39）was selected as the optimal mutant.Secondly,δ-integration system was used to increase ILV3 copies on yeast chromosomes by using plasmid pUC18-δ5’-His-PGK1p-ILV3-δ3’.Meanwhile,plamids YEplac181-TDH3p-Cox4-ARO10-GFP-CYC1,YEplac195-PGK1p-ILV2 and YEplac112-PGK1p-ILV5-GFP-CYC1 were constructed to over-express ARO10,ILV2and ILV5,respectively.Then,we constructed engineered strains W303-1A V2V5V3O10 and EMS39 V2V5V3O10 carrying overexpressed ILV2,ILV3,ILV5 and ARO10.And engineered strain W303-1Aδ5’-His-δ3’YEplac195 YEplac181YEplac112 was used as the control strain.Then,fermentation performances of engineered strains W303-1A V2V5V3O10 and EMS39 V2V5V3O10 were investigated in micro-anaerobic fermentation.And the yields of isobutanol,ethanol and acetic acid were measured GC and HPLC.Our results showed that production of isobutanol in EMS39 V2V5V3O10 were 404.2mg/L and the productivity was 10.11 mg/g glucose,which increased by 5.7-fold and1.5-fold than that in the control strain and that in W303-1A V2V5V3O10,respectively.The production of ethanol in engineered strains W303-1A V2V5V3O10 and EMS39V2V5V3O10 were higher than that in the control strain,but there were no markedly changes in the three strains.The production of ethanol in engineered strain EMS39V2V5V3O10 reached the highest（4.343 g/L）in 32 h.Fermentation performances of engineered strains W303-1A V2V5V3O10 and EMS39V2V5V3O10 in very-high gravity fermentation were also investigated.Our results showed that isobutanol production in engineered strain EMS39 V2V5V3O10 was2.795 g/L in 24 h and the productivity was 69.9 mg/g glucose,which was increased by 1.6-fold than in strain EMS39δ5’-His-δ3’195 181 112.Isobutanol production in engineered strain W303-1A V2V5V3O10 reached the highest（4.206 g/L）in 48 h and the productivity was 100.6 mg/g glucose.And the isobutanol production of engineered strain W303-1A V2V5V3O10 was increased by 4.1-fold than that of the control strain W303-1Aδ5’-His-δ3’195 181 112.Additionally,we constructed plasmids for over-expressing five key genes involved in isoamyl alcohol biosynthesis pathway in S.cerevisiae.These results will lay foundations for further isoamyl alcohol biosynthesis researches.In conclusion,we screened a high isobutanol tolerance mutant（EMS39）by EMS mutagenesis method.And its’isobutanol productivity was improved significantly.These results laid foundations for further improving isobutanol yield in S.cerevisiae. |
