UPRE-lac Z为报告基因评价酵母UPR响应初步研究 *

目的: 未折叠蛋白质反应UPR是酵母最重要蛋白质质量控制机制之一,研究UPR响应规律有助于优化异源蛋白分泌途径合成和应对酸醇等胁迫因子的细胞自我保护。方法: 选择实验室菌株W303-1A和工业菌株An-a,以UPRE启动子控制下的Lac Z为报告基因,利用CRISPR/Cas9技术构建得到指示菌株W303-1A (leu 2::UPRE-lac Z)和An-a (leu 2::UPRE- lac Z),分别简称WZ和AZ。结果: 生长曲线测定显示WZ和AZ与亲本菌株的生长接近;添加下述试剂孵育4h后测定β-半乳糖苷酶酶活:1μg/ml衣霉素、8%(v/v)乙醇、0.3%(v/v)乙酸、5%(v/v)乙醇+0.1%(v/v)乙酸;菌株AZ的比酶活分别是对照值的8.2、26.4、1.1和7.9倍,而菌株WZ则分别为12.6、2.4、1.0和1.0倍;进一步以YEplac195为载体表达β-葡萄糖苷酶,AZ和WZ转化子在2%纤维二糖中生长24h的β-葡萄糖苷酶酶活值分别为0.35和6.12U/ml,相应的LacZ则分别为对照值的3.1和5.4倍。结论: 两个菌株显示了在抑制物和异源蛋白表达UPR响应和调控能力上的显著差异,为其改造利用提供了方向;研究也为分析抑制物耐受性和异源蛋白表达关键制约因素、优化酵母ER和UPR信号通路的调控奠定了初步方法基础。

Evaluation of UPR Response in Yeast by Using UPRE-lac Z as a Reporter Gene

Objective: Unfolded Protein Response (UPR) is one of the most important protein quality control mechanisms, the information about UPR with help to optimize heterologous protein synthesis by secretory pathway and cell self protection from inhibitory substances such as acid and ethanol.Methods: The indicating strains, W303- 1A (leu 2::UPRE-lac Z) and An-a (leu 2::UPRE-lac Z), abbreviated as WZ and AZ, respectively, were first constructed by CRISPR/Cas9 technology and using UPRE-lac Z as a reporter.Results: The growth curve test showed that there was no significant difference between two strains and their host ones. The β-galactosidase activity was assayed at 4 h of incubation time after adding four reagents into media as followings, 1 μg/ml tunicamycin, 8%(v/v) ethanol, 0.3%(v/v) acetic acid, 5% (v/v) ethanol + 0.1%(v/v)acetic acid, respectively. The values of strain AZ were 8.2, 26.4, 1.1 and 7.9 times of that of blank control, respectively, while the results of strain WZ were 12.6, 2.4, 1.0 and 1.0 times, respectively. Further β-glucosidase was expressed by YEplac195 vector into strain AZ and WZ cell. The assay results after 24 h cultivation in 2% cellobiose media showed that the β-glucosidase activities were 0.35 and 6.12 U/ml, respectively, and the responding LacZ values were 3.1 and 5.4 times of that of blank control, respectively.Conclusions: There exists significant difference on the UPR response spectrum between two strains, which also implies their divergent direction and strategy of genetic modification. This study lays the foundation of analytical methods for investigating the key limiting factors in cell inhibitor tolerance and heterologous protein expression in order to make better use of yeast ER and UPR pathway engineering.