YPS1/YPS2失活改进酿酒酵母An-α菌株β-葡萄糖苷酶分泌表达  

Improved β-glucosidase expressing in Saccharomyces cerevisiae An-α by YPS1/YPS2 gene inactivation

作　　者：郭敬涵 陆海燕 洪解放[2] 贾玉蝶 邹少兰[1,2] GUO Jinghan;LU Haiyan;HONG Jiefang;JIA Yudie;ZOU Shaolan(College of Chemical Engineering,Tianjin University,Tianjin 300085,China;Tianjin R&D Center for Petrochemical Technology,Tianjin University,Tianjin 300072,China)

机构地区：[1]天津大学化工学院,天津300085 [2]天津大学石化中心,天津300072

出　　处：《微生物学通报》2021年第12期4485-4495,共11页Microbiology China

基　　金：天津市科技计划(18YFZCNC01240);国家自然科学基金(31470208);国家重点研发计划(2019YFA0905600)。

摘　　要：【背景】工业酵母菌株的蛋白质表达通常存在表达量低、分泌效率低的问题。【目的】考察失活Yapsin蛋白酶Yps1p和Yps2p对β-葡萄糖苷酶在酿酒酵母An-α菌株中表达的影响。【方法】利用CRISPR/Cas9基因组编辑技术,首先构建得到未折叠蛋白响应(Unfolded Protein Response,UPR)指示菌株An-α(leu2::UPRE-lac Z)即An-αL,然后分别失活其YPS1和YPS2基因,导入以YEplac195为载体的β-葡萄糖苷酶表达质粒(简称BG),进行生长和酶活分析评价。【结果】菌株An-αL的YPS1和YPS2基因失活对其在酵母浸出粉胨葡萄糖(Yeast Extract Peptone Dextrose,YPD)培养基中的生长未造成明显的不利影响;导入质粒BG后将在酵母浸出粉胨纤维二糖(Yeast Extract Peptone Cellobiose,YPC)培养基中生长的最大OD_(600)分别提高了21.9%和7.4%;最大总酶活值为0.087 5和0.068 6 U/(m L·OD_(600)),是对照菌株相应值的2.268倍和1.778倍;分泌比例提高了19.4%和22.2%;β-葡萄糖苷酶表达水平与β-半乳糖苷酶酶活水平所代表的UPR信号响应值之间呈现良好的相关性。【结论】YPS1和YPS2基因失活有助于改进酿酒酵母An-α菌株中β-葡萄糖苷酶的分泌表达。[Background]Low level expression and poor secretion efficiency are the common problems in the production of secretory recombinant proteins by industrial yeast.[Objective]To investigate the effect of inactivating yapsin proteases Yps1p and Yps2p on β-glucosidase expression in Saccharomyces cerevisiae An-α,a yeast strain isolated from Angel industrial yeasts.[Methods]By utilizing CRISPR/Cas9-based gene editing technology,we firstly constructed a UPR-indicator strain An-α(leu2::UPRE-lac Z),designated as An-αL.Next,the YPS1 and YPS2 genes were inactivated,followed by transformation of the β-glucosidase-expressing plasmid BG that was constructed on the YEplac195 vector.The growth and enzymatic activities of the resultant strains were evaluated.[Results]Inactivation of the YPS1 or YPS2 gene of the strain An-αL did not affect the cell growth in YPD medium.The presence of the plasmid BG increased maximum OD_(600) values in the YPC medium by 21.9% and 7.4%,respectively.The maximum enzymatic activities were 0.087 5 and 0.068 6 U/(mL·OD600),which were 2.268 and 1.778 times of the control value,respectively.The ratio of the secreted protein was also increased by 19.4% and 22.2%,respectively.There was a good correlation between the β-glucosidase activity level and the UPR signal response value as represented by the β-galactosidase activity level.[Conclusion]Inactivation of YPS1 and YPS2 could improve the secretory β-glucosidase yield of the strain An-α.

关 键 词：YPS1/YPS2基因失活 酿酒酵母An-α Β-葡萄糖苷酶 分泌表达 

分 类 号：Q78[生物学—分子生物学]