Construction Of Recombinant Saccharomyces Cerevisiae By Directed Evolution Of Global Transcription Factor

Global transcription mechanism engineering(gTME)is a technique to optimize cell phenotypes by gene rearrangement,through the modification of global transcriptional regulators to direct selectionof the objective product or target phenotypethat can make changes in the whole transcriptional regulation process to alter or enhance the transcription and expression of target genes,this means can obtain the metabolic flux enhancement or specific phenotype enhanced strain.The transcription factor spt15 belongs to the transcription initiation complex,that is a TATA binding protein,its mutation determines the specificity of the promoter,that can obtain the overexpression ofrelated genes.In this study,gTME was used to construct the core transcription factor spt15,spt3 of Saccharomyces cerevisiae,code gene mutation library and construct the recombinant strain,through the filtering of recombinant strain,canobtain higher ethanol yield and improve the stress tolerance of other yeast strains.If these strains were transformed into industrial yeasts,it can produce far-reaching significance for the development of biomass energy.1.In this paper,according to the spt15 sequences of Saccharo-myces cerevisiae thathave been established,designdegenerate primers,and clone the genes related to wild type yeast strains for industryal fermentation by PCR to construct the mutant library.Finally,12 si-ngle point mutation genes were obtained by sequencing.The 12 point mutationand wild type gene spt15,spt3 and resistance gene G418 are connected to the control of gene expressionvector pYES2 NT,thenconstruct the recombinant plasmid,transforming the plasmid into Saccharomyces cerevisiaeYPH499 by lithium acetate method,this method can obtain a total of 15 recombinant Saccharomyces cerevisiae strains.2.The recombinant yeast strains were screened by setting the tolerance condition,then through the ferment experiment to obtain comparative test of tolerance fermentation between the screened strains and the control strains,and to verify the performance of the screening strains.Tolerance screening results were as follows:INVSC1-spt15-G and INVSC1-spt15-Q can tolerate acetic acid of potency is 6 grams per liter,its tolerance is 3.64 and 4.79 times as much as the control group INVSC1-Neorespectively;INVSC1-spt15-G and INVSC1-spt15-S can tolerate sodium chloride of mass fraction is nine percent,at the same tiame,its tolerance is 1.16 and 1.75 times as much as the control group respectively;INVSC1-spt15-I and INVSC1-spt15-Mcan tolerate the maximum temperature is 43 degrees,its tolerance is 1.58 and 1.45 times as much as the control group INVSC1-Neorespectively;at the same time,INVSC1-spt15-G ? INVSC1-spt15-S and INVSC1-spt15-D can tolerate ethyl alcohol of volume fraction is sixteen percent,its tolerance is 2.76 and 4.17 times as much as the control group INVSC1-Neo respectively.3.After culturing on the seed mediue ofrecombinant strain and the control strain,they were put in 100 ml fermentation medium respectively,the condition is thirty degrees centigrades,two hundred rpm of anaerobic fermentation.After the fermentation of 48 h,measuring the concentration of glucose and ethanol.The results show that:both the control and mutant recombinant strains consumed glucose completely within several hours,the rate of glucose consumption did not affect ethanol production.The ethanol yield of recombinant bacteria INVSC1-spt15-M and INVSC1-spt15-N was greatly improved,the ethanol yield on glucose medium was 43.0±0.9 grams per liter and 43.7±0.2 grams per liter,theyincreased seventeen point eight percent and nineteen point seven percent than the control strain respectively.Under the condition of industrial production of liquefied mash,the recombinant strain and the control strain fermentat 64 h,the recombinant strain INVSC1-spt15-Methanol yield increased significantly,increasing fifteen point six percent than the control strain,the fermentation data was comparised with dextrose culture-medium shows that recombinant strain INVSC1-spt15-M ethanol yield all increased than the control strain.4.The result of comparison of strains with high ethanol yield and improved toleranceshows that:the improvement of tolerance does not necessarily lead to the improvement of fermentation efficiency,tolerance and ethanol fermentation efficiency are controlled by different genes.It was also found that the over expression spt15?spt3 gene had no obvious effect on the tolerance and ethanol production of the strain.5.Through the preliminary study on the sequencing of recombinant strains that the ethanol yield was increased observably,obtaining new transcription factor mutation genes,two mutant genes were mutated from lysine to methionine and asparagine in 127 placesrespectively.The results revealed that the effective mutation of spt15 gene caused significant changes in metabolic pathway and metabolic flux of Saccharomyces cerevisiae.