Improving Ethanol Tolerance Of Saccharomyces Cerevisiae By Directed Evolution Of SPT8

Ethanol tolerance is an important property of Saccharomyces cerevisiae for bioethanol fermentation.If we can improve the ethanol tolerance of saccharomyces cerevisiae,it would be to improve the fermentation performance and reduce the production cost,which is of great significance,it can promote the development of biological ethanol production.Here we report the ethanol tolerance of Saccharomyces cerevisiae from the transcriptional level by engineering its global transcription factor SPT8,which is an important subunit of transcription complex SAGA.In the process of transcription,the combination of SAGA and TBP by SPT8.The results were described as follows:1.The strong PGK promoter、CYCI terminate and SPT8 fragment were inserted into plasmid pUG6 to obtain expression vector pUPST.And the vector was transformed to Y01 for construction of transgenic bacteria YS1.Statistical analysis showed that the OD600 of YS1 and Y01 were no obvious difference(P<0.05).2.A mutant gene library of SPT8 was constructed using error-prone PCR,and the library was transformed to yeast strain Y01.Mutant YSEpl and YSEp2 were selected with improved ethanol tolerance from the library.It was found that the OD600 of YSEpl and YSEp2 growing in presence of 14%ethanol in YPD medium for 64 h were higher than the control strain,and the highest cell densities of YSEpl and YSEp2 were increased by 8.94%(P<0.01)and 4.67%(P<0.01),respectively.But,with no significant difference in the growth rate in YPD medium without ethanol.In the same fermentation condition,the ethanol yield in YSEpl and YSEp2 could reach 10.8%(P<0.01)and 4.81%(P<0.01)of that of control.3.For the biological safety and no harmful antibiotic resistance of engineering strains,KanMX resistance gene was knocked out.After knocking out KanMX,the strains were named YSEpl-K and YSEp2-K.By protein sequence analysis of the mutant SPT8 from YSEp1-K and YSEp2-K showed that the same mutations of two mutant strains were Asn156His and Gly585Ser,which may responsible for the interaction of SPT8 with TBP,and then may be responsible for the improvement of ethanol tolerance.4.Through determination of SPT8 expression by qRT-PCR and the related physicochemical analysis,it was concluded that the YS1,YSEpl-K and YSEp2-K of SPT8 gene mRNA expression levels were 3.405-fold(P<0.01),5.312-fold(P<0.01)and 5.312-fold(P<0.01)of the original strain Y01.