Study On Improving Tolerance To Isobutanol By Global Transcription Machinery Engineering In Saccharomyces Cerevisiae

For a long time,the demand for energy has been increasing.The production of alcoholic biofuels by fermentation using microorganisms as cell factories has attracted extensive attention of scientists all over the world.Isobutanol is recognized as one of the second-generation alcohols biofuels.The synthesis of isobutanol by Saccharomyces cerevisiae(S.cerevisiae)has high market value and broad application prospects.However,S.cerevisiae cells are sensitive to high temperature and osmotic stress in fermentation processes.In addition,isobutanol is toxic to yeast cells,which would directly affect the improvement of isobutanol production.Therefore,it is important to enhance tolerance to isobutanol of S.cerevisiae and optimize the ability of cells to produce isobutanol.In this work,the global transcription machinery engineering(g TME)method was used to screen strains with higher tolerance to isobutanol.Firstly,the spt15 mutant gene library that was about 4×10⁶in size was constructed by error-prone PCR.Then the plasmids that contain spt15 were transferred to W303-1A,and about 5×10⁵transformants were obtained.Transformants were randomly selected and propagated on SC minus tryptophan medium with 4 g/L to 8 g/L isobutanol for primary selection.55 strains were selected out,and growths of these mutant strains were determined on SC minus tryptophan and SC minus tryptophan with 12 g/L isobutanol by dilution.The strains spt15-3 and spt15-43 were screened by this method.Plasmids YCplac22-spt15-3 and YCplac22-spt15-43 in strains spt15-3 and spt15-43 were extracted and retransformed into wild type strain W303-1A,and their tolerance to isobutanol were further verified.Our results indicated that the strain carrying YCplac22-spt15-3(named mutant spt15-3)grew well on SC minus tryptophan medium with 12/L isobutanol and could grow on SC minus tryptophan medium with 16g/L isobutanol.And there was no significant difference between growth of the strain carrying YCplac22-spt15-43(named mutant spt15-43)and the wild-type strain on SC minus tryptophan medium with 12 g/L isobutanol.But growth of mutant spt15-43 and the wild-type were significantly lower than that of mutant spt15-3.And mutant spt15-43 and the wild-type could not survive on SC minus tryptophan medium with 16 g/L isobutanol.The mutations loci of SPT15 gene on plasmids YCplac22-spt15-3 and YCplac22-spt15-43were determined by sequencing.The results showed that there were 2 mutants loci in the promoter and 1 synonymous mutation in the open reading frame of SPT15 gene in plasmid YCplac22-spt15-3,and there was 1 mutant locus in the open reading frame of SPT15 gene in YCplac22-spt15-43.Finally,the isobutanol tolerance of mutant spt15-3 was determined by survival experiment.Our results suggested that the survival rate of strain spt15-3 under20 g/L isobutanol pressure was twice times more than that of control(W303-1A containing plasmid YCplac22),1.3 times more than that of converting natural SPT15 genes(strain Control-1),respectively.The lethal concentration isobutanol of mutant spt15-3 was 24 g/L.The fermentation property of mutant spt15-3 was tested by fermentation.Firstly,optimization of valine metabolism pathway was carried out in S.cerevisiae.Gene ILV2,ILV3 and ARO10(encoding acetolactate synthase,dihydroxy acid dehydrates and ketoacid decarboxylase in valine metabolic pathway,respectively)were overexpressed.Compared with that of the control strains Ctrl-1,isobutanol yield of strains Nor-2 increased by 3.06times.Secondly,the micro-anaerobic fermentation conditions of yeast were optimized.The initial OD_600nmwas increased from 0.5 to 3.5,and the initial glucose concentration was increased from 25 g/L to 65 g/L.The fermentation results showed that the growth rate and sugar consumption rate of cells were accelerated and cell concentration was increased.The isobutanol productions of Nor-3,Engi-4 and Engi-5 were 0.52 g/L,0.45 g/L and 0.42/L,increased by 7.53 times,6.45 times and 6.00 times compared with the Control-1,respectively.And Nor-3,Engi-4 and Engi-5 generated highest isobutanol yield at 28h,which were 8.36 mg/g glucose,7.29 mg/g glucose and 6.78 mg/g glucose,respectively.They were 9.5 times,8.3 times and 7.7 times compared with Ctrl-1,respectively.Under the fermentation conditions of high cell inoculation and high initial glucose concentration(OD_600nmwas 3.5 and glucose concentration was 65g/L),isobutanol yields of strain Ctrl-1,Nor-2 and Nor-3 decreased after 24h.While yields of Engi-4 and Engi-5 were maintained7.15 mg/g glucose and 6.48 mg/g glucose during the whole fermentation process.In addition,the effects of overexpressing gene OLE1(encoding?-9 desaturase),gene CWP2(encoding cell wall proteins)and gene AQY1(encoding water transporting aquaporin)on tolerance to isobutanol in yeast were studied.Firstly,plasmids YEplac181-PGK1p-OLE1,YEplac195-CWP2 and YEplac181-PGK1p-AQY1 were constructed.Then,plasmids YEplac181-PGK1p-OLE1,YEplac195-CWP2 and YEplac181-PGK1p-AQY1 were transformed into W303-1A,designated as strains W303-1A-OLE1,strains W303-1A-CWP2 and strains W303-1A-AQY1,respectively.Finally,the tolerance to isobutanol of strains W303-1A-OLE1,W303-1A-CWP2 and W303-1A-AQY1 were tested.