| Proteinase A(PrA)is encoded by PEP4-allele and is required by the Saccharomyces cerevisiae vacuolar proteolytic system during nutritional stress,sporulation,and vegetative growth circumstances.This study on the effect of PEP4-alleles on acid stress tolerance of an industrial yeast S.cerevisiae strain was done to construct one PEP4-allele disrupted industrial S.cerevisiae strain and modify another PEP4-allele of the derived strain with the constructed DNA fragment.This study investigated the impact of PEP4-alleles on industrial yeast S.cerevisiae cellular tolerance and cellular growth under citric acidic stress.In addition,this study conducted a preliminary investigation on the path of intracellular PrA activity on cellular citric acid stress tolerance regulatory mechanism in industrial S.cerevisiae strain.The wild-type industrial S.cerevisiae strain SWY85 was used as the starting strain to derive the strain SWY85F(with one PEP4-allele disrupted)and strain SWY85S(with second PEP4-allele modified).For screening of the industrial S.cerevisiae transformants carrying the Creexpression plasmid,we endowed the Sh ble coding sequence to the plasmid p SH47 to construct the plasmid p SH/Zeo,and transformed the constructed plasmid to the recombinants with one PEP4-allele disrupted,and induced the expression of Cre-recombinase to rescue the Kan MX screening marker to serve the second PEP4-allele modified transformants screening and induced the expression.To ensure the homologous recombination site is not the same on the one PEP4-allele,we inserted the DNA fragment Kan MX to construct the plasmid p HR31,using the constructed plasmid as template,the DNA fragment amplified by PCR was transformed into yeast SWY85 F,and the recombinant strain was confirmed by colony PCR.The citric acid stress tolerance and growth performance of the two strains SWY85 F and SWY85 S were experimented and the results showed that the second PEP4-allele modification significantly increased the intracellular PrA activity.The cellular tolerance of the strains under citric acid stress was compared,and the cellular tolerance of the strain with one PEP4-allele disruption(SWY85F)was significantly reduced while the second PEP4-allele modification significantly increased the strain SWY85 resistance against citric acid stress.The second PEP4-allele modified strain(SW8Y5S)increased the intracellular trehalose content.It was found that the produced intracellular protein concentration was regulated by the second PEP4-allele modification. |
