Study On The Effect Of Key Gene Modifications In Protein Secretion Pathway On β-glucosidase Expression In Yeast

Saccharomyces cerevisiae is used in large-scale alcohol production.Industrial yeast strains are highly robust to all kinds of stresses and have perfect alcohol-producing performance.However,their protein secretory capacity would limit the potential to be used as a platform for biopharmaceutical production and enzyme preparation.In this study,β-glucosidase,one of the most important components in enzymatic hydrolysis and utilization of lignocellulosic,was used as a marker to study the effect of the modification of key genes in protein secretory pathway on the exogenous protein expression,which will lay the basis forβ-glucosidase expression with high level and high anchoring or secreting efficiency.Therefore,five key genes of yeast protein secretory pathway were selected to be inactivated or overexpressed to investigate the effect on the anchoring or secretion expression ofβ-glucosidase.Details are as follows:Ⅰ:Effect of single gene modification onβ-glucosidase expression1.Inactivation of YPS1 and YPS2The fermentation supernatants from the strain AαL（YPS1）（BG）,AαL（YPS2）（BG）and the control AαL（BG）were collected,and protein concentration analysis and SDS-PAGE were carried out.The difference of the protein banding pattern between the strains indicated that theβ-glucosidase expressed was partly degradaed,and the YPS1or YPS2 gene inactivation significantly increased theβ-glucosidase content.2.Overexpression of ERV25、PDI1、KAR2 genesThe three strains,AαL（ERV25）,AαL（PDI1）and AαL（KAR2）,were constructed by CRISPR/Cas9 gene editing technology,respectively.Further,the anchoring expression plasmid BG-cwp and the secreting expression plasmid BG were introduced,respectively.The growth,enzyme activity and fermentation of the resultant strains and the control strain were evaluated in YPC.The results showed:1)Growth and cellobiose:ERV25 gene overexpression promoted the growth and cellobiose consumption.The cellobiose was depleted by strain AαL（ERV25）（BG-cwp）and AαL（ERV25）（BG）within 36-48 h and about 24 h,respectively,and therefore they entered the stastionary phase earlier than other strains;2)Alcohol production:The overexpression of three genes increased the maximum ethanol concentration from cellobiose,and ERV25 overexpression led the highest titers,that is,the maximum values from strain AαL（ERV25）（BG-cwp）and AαL（ERV25）（BG）were 7.797 g/L and8.669 g/L,respectively,while the corresponding values of the control strains were5.698 g/L and 5.998 g/L,respectively;3)Enzyme activity:host gene modification increased the maximum enzyme activity in varying degrees,the maximum values were obtained at 72 h for anchoring strains and at 48 h for secretory strain.While the anchoring ratio kept constant or was slightly decreased,the secretion ratio of secretory expression strains increased significantly by 22%to 36%.4)UPR response:The UPR response value represented byβ-galactosidase activity was positively correlated withβ-glucosidase activity.In conclusion,the modification of the three genes could effectively promote the expression ofβ-glucosidase in yeast,and the effect ranged from large to small in order of ERV25,KAR2 and PDI1 gene.Ⅱ:Effect of double gene modification onβ-glucosidase expressionAαL（ERV25/yps1）,AαL（yps1/yps2）,AαL（KAR2/PDI1）,AαL（PDI1/yps1）and AαL（KAR2/yps1）were constructed in the same way,and were further introduced by plasmid BG-cwp and BG,respectively.The results from evaluating experiments in YPC showed:1)Growth and cellobiose:All strains entered the stationary phase at about 84 h,and cellobiose was depleted in 36-48h.Among them,the ERV25/YPS1and YPS1/YPS2 strains were showed to have more advantages on growth than others.2)Enzyme activity:The enzyme activity of all strains was higher than that of the control strains except AαL（PDI1/yps1）（BG-cwp）,which had the maximum value0.0703 U/m L/OD_600.The activity values in secretory expression mode were generally higher than those in anchoring expression mode.Among all strains,AαL（ERV25/yps1）（BG）and AαL（yps1/yps2）（BG）showed highest activities,0.5460and 0.4746 U/m L/OD₆₀₀at 48 h,respectively,which were 3.37 and 2.67 times of the corresponding values of control strains.But for anchor-expressing strains,the highest value 0.2158 U/m L/OD₆₀₀was obtained by AαL（yps1/yps2）（BG-cwp）at 48 h.While yps1/yps2 inactivation had no obvious effect on anchoring ratio in yeast with anchoring expression way,other double gene modifications all decreased the anchoring ratio.However,the ratios of supernatant activity were increased to varying degrees in all strains with secretory expression way,and the highest value by AαL（ERV25/yps1）（BG）was 1.61 times higher than that of the control strain.In conclusion,ERV25/yps1 and yps1/yps2 were showed to be the best double modification to promoteβ-glucosidase expression in yeast,while PDI1/yps1 was not suitable for anchoring expression mode because of unknown causes.Ⅲ:Evaluation of plasmid stabilityThe stability of three plasmids YCplac33、YEplac195 and YEplac195-Aa BGL（abbreviated as BG）in strain AαL and AαL（yps1/yps2）were evaluated.The results showed that yps1/yps2 dual inactivation significantly improved plasmid stability,while theβ-glucosidase expression significantly decreased plasmid stability.The above results will help to improve protein production in yeast,to deepen the understanding on protein secretion pathway and its regulation in industrial strain,so as to better develop and utilize this important industrial microorganism.