Study On Cellulase Expression In Saccharomyces Cerevisiae: The Influencing Factors And Its Application

Using engineering of S.cerevisiae to efficiently express cellulase and produce ethanol from cellulose would beneficial to reduce production costs.This study aim to reveal the key influence factors of secretory or display expression cellulolytic enzyme in S.cerevisiae,and obtain the recombinant strains with efficiently expressing cellulase.The thesis had conducted systematical research to the effect of several factors,such as,the type of anchored peptide sequences,host background,the existence form of gene,gene copy number,culture conditions,on the expression of TrEG?or AaBGL gene.The key factors of influence the expression and optimal way of efficient expression were identified.Finally,Using ? sequence as integration sites,the optimal expressing cassette for the ?-glucosidase gene from Aspergillus aculeatus was integrated into the chromosome of industry S.cerevisiae.Based on these,to investigate its potential application as CBP microbe,the recombinant strains were comprehensively evaluated.Study on the display expression of TrEG?geneThe growth and enzyme activity of expression were evaluated in twenty-four kinds of engineering strains,which were constructed by the prophase work and the work,and the results indicated that:(1)Anchored peptide sequences and its type had significant impact on the distribution of TrEG?enzyme activity.The anchored effect of three anchored peptide sequences(Cwp2,Linker-Cwp2,Ag?)was increasing sequentially,and Ag? anchored peptide had the best anchored effect in the same type strains,and the anchored ratio of Ag? can reach 86.3%.(2)The type of anchored peptide sequences had significant effects on expression of enzyme activity.The EW3 strains expressing Cwp2 fusion protein had the highest total enzyme activity,which can reach 1.20 U/ml/OD660.(3)The enzyme activity of display expression was significantly lower than that of the secretory expression,and the latter can reach 1.784 U/ml/OD660.(3)Improving the gene copy number can significantly increase the expression of TrEG?enzyme activity,but had no significant effect on the proportion which the cell enzyme activity accounts for the total enzyme activity.Study on the display expression of AaBGL geneThe growth and enzyme activity of expression were evaluated in five kinds of engineering strains,the results indicated that:(1)The type of anchored peptide sequences had significant impact on the distribution of AaBGL enzyme activity.The anchored ratio of strains containing Ag? or Cwp2 fusion proteins nearly the same is about 93%.(2)The type of anchored peptide sequences had significant effects on the expression of enzyme activity.The strain containing Cwp2 fusion protein had the highest total enzyme activity,and total enzyme activity reached 0.066 U/ml/OD660.The enzyme activity of display expression had significantly lower than that of the secretory expression,and the latter can reach 0.120 U/ml/OD660.(3)Improving gene copy number can significantly increase the expression of AaBGL enzyme activity,but had no significant effect on the distribution of the enzyme activity.(4)The host background had no significant effect on the enzyme activity.Study on the secretory expression of AaBGL geneUsing the ? integration method to obtain high copy integrated AaBGL engineering strains in the industry S.cerevisiae,the recombinant strains were comprehensively evaluated.The results showed that:(1)the ?-integration way was successfully applied to industrial S.cerevisiae to quickly obtain more copies integrated strain with the efficient and stable exogenous gene expression.(2)The expression of beta-glycosidase enzymes do not affect growth in industrial strains,and the highest enzyme activity can reach 3.19 U/ml.The maximum ethanol concentration of industrial engineering strains were 3.98%,and is higher than Angel yeast in 10% solid content cellulose material medium,with 10 FPU/g Biomass cellulase enzymes,without beta glycosidase enzymes.This study helps to deepen the laws of expression exogenous genes in saccharomyces cerevisiae,and give full play to their potential application in protein production and ethanol fermentation.