| As the total amount of fossil energy in the word is decreasing and human demand is increasing continuously,searching for new alternative energy is a major problem that human beings must solve.Bio-fuel ethanol is widely concerned with its green and renewable characteristics,China has become the third largest producer and application in the world after Brazil and United States.At present,almost all the fuel ethanol produced in the world was produced by the fermentation of Saccharomyces cerevisiae strain,and the high performance of Saccharomyces cerevisiae strain is an important factor in the development of the fuel ethanol industry.In the fermentation process,the ability of Saccharomyces cerevisiae cell fermentation to produce ethanol and the tolerance of various adverse factors in the fermentation environment are important indicators for the application of saccharomyces cerevisiae cell in industry.Therefore,it is a meaningful work to obtain the Saccharomyces cerevisiae strain with enhanced ethanol fermentation capacity and tolerance and to study the mechanism of its high yield and tolerance.High osmotic pressure caused by high concentration of sugar is an environmental stress encountered frequently in the industrial application of Saccharomyces cerevisiae strain.In the early study,the research on the mechanism of high permeability of Saccharomyces cerevisiae was mainly focused on the high permeability environment of salt and sorbitol.There is not much research on the tolerance mechanism of high osmosis formed by sugar.In this study,we got a mutant Saccharomyces cerevisiae strain UV02_HG which had a greater ability to grow and ferment under stress of 40%(W/V)glucose than the wild type strain MF02 which was screened and preserved in my laboratory with high yield of molasses ethanol through UV mutagenesis.The growth curves of the two stains in YPD medium were basically consistent and the average maximum cell number of mutant strain increased from 3.38×108 to 3.81×108.The growth and ethanol fermentation conditions of mutant strain IUV02 HG were superior to wild type strain MF02,the maximum cell number of mutant strain increased from 6.857×108 to 8.767×108,increased 27.85%than the wild type strain,the largest ethanol production rate increased from 14.056%(V/V)to 15.304%(V/V),8.88%higher than that of wild type strain.Transcriptome sequencing was performed on the gene expression of the two strains growing to exponential growth in YP40 medium,as the results shown,compared with the wild type strain,1203 genes were considered to have significantly different expression in mutant strain,among these genes,363 were up-regulated and 840 were down-regulated,and 1846 genes were considered to have significantly SNP calling,269 of them have different expression meanwhile.The KEGG enrichment results show that the differentially expressed genes were found mainly in pathways such as the ribosome,oxidative phosphorylation,protein processing in the ER,phagosome and endocytosis,protein export,sugar metabolism pathway and MAPK pathway.Almost all the genes involved in ribosome,oxidative phosphorylation and protein export were down-regulated,while most of the genes involved in sugar metabolism pathway and MAPK pathway were up-regulated.We got a network diagram which including only one interaction relationship of transcription factor-DNA by PheNetic network analysis.There were 75 genes interact with 77 points in the network,26 genes were got as the trait induced gene and ranked according to the score.20 genes were randomly selected for quantitative fluorescence PCR,the verification results showed that the accuracy of the transcriptom sequencing data was up to 95%.We also detected the cell relative respiration intensity of the two strains under high sugar pressure,the result were consistent with transcriptome sequencing.The relative respiration intensity of mutant strain was just 74.2%of the wild type strain.According to the results of PheNetic analysis,the first two induced genes STE12 and TUP1 were selected to perform single gene knockout in the haploid cells of the mutant strain UV02_HG respectively using Cre/loxp system.After gene knockout,the MATa and MATa type haploid cells were redoubled,and the KanMX resistance was eliminated by pSH65 plasmid to get gene deficient strains UV02_HG?STE12 and UV02_HG?TUP1.In order to further study the effects of two genes on the high glucose tolerance of Saccharomyces cerevisiae,one step method was used to quickly connect TEF promoter,open reading frames and CYC1 terminator to the vector with KanMX resistance,according to the cutting features of BsmBI incision enzyme.The sequences of"TEF promoter-TUP1/STE12 ORF-CYC1 terminator-KanMX" were amplified using primers with homologous sequence,and then integrated into the original position by homologous replacement in haploid cells of gene deficient strains.Haploid cells redoubled to get strains UV02_HG?STE12Hb and UV02_HG ATUP1Hb with recovering of two genes.The character changes of gene knockout and recovering strains were detected,the growth rate of STE12 gene deletion strain UV02_HGASTE12 was slower than UV02_HG strain in the early stage in YPD medium and faster after 12 hours.The tolerance of UV02_HG?STE12 strain was significantly higher than that of UV02_HG strain whether in YP40 liquid medium on solid medium plates including high concentration of glucose.The maximum cell number in YP40 medium was increased 24.2%of UV02 HG strain,while the ethanol production and the tolerance ability to ethanol had no significant changes.Both the cell relative respiration intensity and cell membrane permeability of UV02_HGASTE12 strain were decreased.There was no significant change in the growth rate of UV02_HG?STE12Hb strain in YPD medium,and was slightly decreased in YP40 medium,while the yield of ethanol fermentation having no significant change.Both the relative respiration intensity and cell membrane permeability of protein of strain UV02_HG?STE12Hb improved slightly.GO enrichment of the 16 unknown genes affected by STE12 gene in the network diagram obtained from PheNetic analysis,showed that these proteins are associated with cell membrane under hypertonic environment,they interact with endoplasmic reticulum membrane and cytoplasmic membrane by hydrophobic transmembrane protein,According to the integration of results,we speculated that the STE12 transcription factor plays a role in the tolerance of Saccharomyces cerevisiae cell to high glucose through cell membrane,and it has little effect on the metabolism pathway of ethanol in Saccharomyces cerevisiae cell.Compared with the strain UV02_HG,the growth of TUP1 gene deletion strain UV02_HG?TUP1 was significantly inhibited in YPD medium,both growth rate and maximum cell number were lower than UV02_HG strain,the mean maximum cell number was just 87.7%of UV02_HG strain.However,the growth rate and ethanol yield were higher than that of UV02_HG strain in high glucose medium.The cell relative respiration intensity changed significantly in YPD and YP40 medium,which was less than 20%of UV02 HG strain in YPD medium and increased to 90%in YP40 medium.The growth of TUP1 gene covering strain UV02_HG?TUP1Hb picked up in YPD medium and the relative respirantion intensity increased correspondingly,while the growth rate and ethanol yield reduced in YP40 medium,and cell membrane permeability of nucleic acid decreased too under stress of 40%glucose.According to the results,we speculated that cell respiration regulated by TUP1 gene affect the normal growth of cells,but there were other regulatory factors under stress environment and duiring stress reaction process when TTP1 gene was deleted.TUP1 gene also participate in the regulation of glucose metability and cell membrane permeability,especially in the permeability of nucleic acid,the permeability of nucleic acids increased remarkably when TUP1 gene were deleted and subsided after covering. |
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