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

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-lacZ）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OD₆₀₀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/（mL·OD₆₀₀），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。

Abstract:

[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-lacZ), 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 OD₆₀₀ 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.