?-glucosidase Expressing Optimization And The UPR Response In Industrial Yeast

Saccharomyces cerevisiae is one of the most important ethanol producers 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 the the haploid strain An-?(MAT? ura3)derived from industrial strain and the UPR signal response was studied,which will lay the basis for the final?-glucosidase expression with high host compatibility,high level,and high anchoring or secreting efficiency.The details are as follows:1.The gRNA expressing vectors pRS42H-g LEU2 and pRS42H-g HIS1 were first constructed,and three donor DNA fragments targeting LEU2,HIS1 and LEU2 ORF were prepared by PCR,respectively.The gRNA expression vector and donor DNA fragment were co-transformed into An-? competent cells by lithium acetate method.Then three strains An-?(leu2),An-?(leu2 his1)and An-?(leu2::UPRE-LacZ)were obtained through identification and screening.Thus,a complete set of CRISPR/Cas9 gene editing technology for industrial strain An-? was initially established.The obtained three strains could be used as platform strains,which carries two or three auxotrophic selection markers or UPR response reporter,respectively.2.The plasmids YEplac195-Aa BGL and YEplac195-Aa BGL-cwp2 had previously been constructed in our laboratory for ?-glucosidase expressing by secreting and anchoring,respectively.The promoter Ptpi and the secreted peptide xyn2 s coding sequence of them were replaced by the Sed1 p promoter Psed1 p and the secreted peptide Ssed1 p coding sequence by PCR and seamless recombination,and then YEplac195-PSsed1-Aa BGL and YEplac195-PSsed1-Aa BGL-cwp were constructed;four plasmids were introduced into the strain An-?(leu2::UPRE-LacZ).Comparative evaluation in 2% cellobiose medium showed that PSsed1 increased the extracellular ?-glucosidase activity ratio of secreted expression strain by 20.12%(24 h)and 8.57%(48 h),and the cell enzyme activity ratio of anchored expression strainshowed almost no change.The total enzyme activity value of the two strains decreased and the LacZ enzyme activity level is positively correlated with the ?-glucosidase enzyme activity level as a whole.3.The Yapsin proteinase-encoding genes YPS1 and YPS2 were inactivated by the forementioned procedure to lead to strains An-?(leu2::UPRE-LacZ)(yps1)and An-?(leu2::UPRE-LacZ)(yps2),which are abbreviated as A?L(yps1)and A?L(yps2),respectively.The growth evaluation in YPD liquid medium initially showed that gene inactivation caused no significant difference from the host strain.The plasmids YEplac195-Aa BGL and YEplac195-Aa BGL-cwp2 were further introduced into the two strains.Comparative evaluation in 2% cellobiose medium showed that the inactivation of YPS1 and YPS2 genes significantly increased the total ?-glucosidase activity and promoted the cellobiose utilization.It also increased the anchoring ratio of anchored strain and secretion ratio of secreted strain in different degrees.The 38 h enzyme activities of strains A?L(yps1)(BG-cwp)and A?L(yps2)(BG-cwp)were2.077 and 1.731 times of that of the control strain A?L(BG-cwp,respectively,and the anchoring ratio increased by 2.50% and 3.11%,respectively.At the same time,the 38 h enzyme activities of A?L(yps1)(BG)and A?L(yps2)(BG)were 2.268 and 1.778 times of that of the control strain A?L(BG),respectively,and the secretion ratio increased by 19.4% and 22.2% respectively.There was a good correlation between the ?-glucosidase activity level and the UPR signal response value represented by the?-galactosidase activity level.