Directed Evolution Of SPT3 To Improve Ethanol Tolerance Of Saccharomyces Cerevisiae

Ethanol tolerance is an important property of Saccharomyces cerevisiae strains for fuel ethanol fermentation.Improvement of ethanol tolerance of yeast cells is beneficial for fuel ethanol production,especially for Very High Gravity(VHG) ethanol fermentation.However, yeast ethanol tolerance is controlled by multiple genes,and is difficult to be efficiently enhanced by single-gene manipulations.In the previous studies,global Transcription Machinery Engineering(gTME) technique has been developed to manipulate SPT15,which encodes one of the components for yeast transcription complex TFIID,and yeast mutant with improved ethanol tolerance was isolated.The alternation of SPT15 interaction with SPT3, which is the component of SAGA transcription complex,was proposed to be responsible for the improved ethanol tolerance in the yeast mutant.In this study,effect of directed evolution of SPT3 on yeast ethanol tolerance was explored.Expression plasmid with the constitutive expressed SPT3 under the strong PGK promoter based on pYES2.0 was constructed.Mutant gene library of SPT3 was constructed using error-prone PCR,and the library was transformed to yeast strain ATCC4126.Mutant M25 was selected with improved ethanol tolerance from the library.The OD₆₀₀ of M25 growing in presence of 10%ethanol in YPD media for 36 h was 2.6 fold of the control strain,and the highest cell density of M25 was 1.2 fold of that of control,with no significant difference in the growth rate in YPD medium without ethanol. The final ethanol concentration of M25 was improved 12.5%of that of control using 125 g/L glucose for ethanol fermentation.Protein sequence analysis of the mutant SPT3 from M25 mutant strain showed that the Ile⁵⁹Thr mutation in the N-terminus conserved region,which is responsible for the interaction of SPT3 with TBP,may be responsible for the improvement of ethanol tolerance in M25.In the two yeast mutants(M1,M2) with decreased ethanol tolerance, the growth rate in the growth medium without ethanol was also decreased.SPT3 mutant protein in M1 was found to have 85 amino acid tnmcation in the C-terminus,while in M2,mutations in Ala 249 and 320 Trp in C-terminus conserved region were revealed.No significant change in ethanol tolerance,growth rate and fermentation rate was found in yeast mutant with the overexpression of SPT3.The results in this thesis showed that SPT3 can be used as another target for improvement of yeast ethanol tolerance.Optimization of mutation efficiency,promoter strength as well as vector copy number will yield yeast mutants with further improvement in ethanol tolerance and ethanol fermentation efficiency.This work provides basis for both the enhancement of ethanol tolerance in industrial yeast strains,and improvement of the economical and technological standards of fuel ethanol fermentation.