?-glucosidase Displaying And Secreting Expression And The UPR Response In Yeast

Saccharomyces cerevisiae is one of the most important ethanol producer for the large-scale alcohol industry,and now it has also become an increasingly important platform for biopharmaceutical production and enzyme preparation.All kinds of genetic modification and utilization of S.cerevisiae depend on protein expression and regulation.In this study,?-glucosidase,one of the most important components of enzymes in lignocellulose digestion and utilization,was expressed by displaying and secreting mode in yeast and the UPR signal response was studied;Thus,their difference on resultantmetabolic burden and protein quality control mechanism were investigated,which will lay the basis for the final?-glucosidase expression with high host compatibility,high level,and high anchorage efficiency.In this study,the haploid strain An-a derived from industrial strain and laboratory haploid strain W303-1A in our lab were selected as the host strains.To first test the effect of CRISPR-Cas9 gene editing technique on the two strains,their HIS1 gene-deficient strains were attempted to be constructed.So the Cas9 expression vector,YCplac33-Cas9,and the gRNA expression vector,pRS-gRNA-HIS1,were first constructed,and donor DNA fragments were prepared by PCR.Then the strains An-a??his1?and W303-1A??his1?were successfully constructed at one time.The recombination efficiencies were 4.4 and 39.8 cfu/pmol donor DNA,and the positive ratio were 74.4%and 98.0%,respectively.The gRNA expression vector pRS-gRNA-LEU2 targeting LEU2 gene was further constructed,and the donor DNA fragment containing UPRE-LacZ gene expression cassette was also prepared by PCR.The An-a?leu2::UPRE-LacZ?and W303-1A?leu2-3::UPRE-LacZ?,being UPR response indicator strains,were obtained at one time according to the procedure mentioned above.The recombination efficiencies were 3.36×10² and 3.46×10² cfu/pmol donor DNA,and the positive ratio were 41.8%and 25.4%,respectively.So the CRISPR/Cas9 gene editing technology procedure introduced was proved to truly be efficient.The results of strain An-a?leu2::UPRE-LacZ?and W303-1A?leu2-3::UPRE-LacZ?evaluation showed that:1)They could grow well in YPD medium similar to that of their parent strain,which implied that the manipulation procedure had no significant negative effect on the growth of the recombinant strains.2)Among the four treatments,1?g/mL tunicamycin,8%?v/v?ethanol,0.3%?v/v?acetic acid,5%?v/v?ethanol+0.1%?v/v?acetic acid,the strain An-a?leu 2::UPRE-LacZ?showed the strongest UPR response to 8%?v/v?ethanol and W303-1A?leu 2-3::UPRE-LacZ?to 1?g/mL tunicamycin.The LacZ enzyme activities were 26.79-fold and 12.68-fold of the corresponding control values,respectively,which showed that their UPR signal response spectra greatly differed from each other.The plasmids YEplac195-Aa BGL-cwp2 with?-glucosidase-displaying expression and YEplac195-Aa BGL with secreting expression mode were both introduced into the above indicator strains.The results of the growth,fermentation,enzyme activity and signal response evaluating for four strains AZ?BG-cwp2?,AZ?BG?,WZ?BG-cwp2?and WZ?BG?,clearly indicated the extra burden of anchoring over secreting expression of?-glucosidase.The strains with cwp2 anchoring peptide led to lower enzyme activity,growth rate,maximum OD₆₀₀00 value and sugar-utilizing and alcohol-producing rate.On the other hand,the degree to be decreased and affected depended on the host genetic background and the oxygen supply condition.Thereby,WZ?BG?and WZ?BG-cwp2?were showed to produce extremely higher enzyme activity than that of AZ?BG?and AZ?BG-cwp2?,11.01,4.65,1.04and 0.83 U/ml,respectively,after 72 h aerobic culture,and higher glucose consumption,growth and alcohol production rates.While AZ?BG?and AZ?BG-cwp2?entered into stationary phase at the same time,the maximum OD₆₀₀00 values obtained by AZ?BG-cwp2?were 68.8%,91.4%,and 71.0%of that by AZ?BG?,respectively,which demonstrated obvious inhibition effect.On the other hand,the UPR signal response value decreased by the strain turn,WZ?BG?,WZ?BG-cwp2?,AZ?BG?and AZ?BG-cwp2?in all three cases,with the highest values being 157.52,91.88,15.63 and 15.04 U/mg proteins,respectively.This implied that under the above conditions,there is a good relationship between the UPR signal response and protein expression level.The analysis that which factor lead to the more significant burden of displaying over secreting in industrial strains is still in progress.