<i>UPRE-lac</i> Z为报告基因评价酵母UPR响应初步研究 <sup>*</sup>

doi:10.13523/j.cb.2006036

中国生物工程杂志

2020, Vol. 40

Issue (10): 1-9 DOI: 10.13523/j.cb.2006036

研究报告

UPRE-lac Z为报告基因评价酵母UPR响应初步研究 ^*

章小毛¹,郭敬涵¹,洪解放²,陆海燕¹,丁娟娟¹,邹少兰^1,^***(

),范寰^3,^***(

)

1 天津大学化工学院系统生物工程教育部重点实验室天津 300072
2 天津大学石化中心天津 300072
3 天津市畜牧兽医研究所天津 300381

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

ZHANG Xiao-mao¹,GUO Jing-han¹,HONG Jie-fang²,LU Hai-yan¹,DING Juan-juan¹,ZOU Shao-lan^1,^***(

),FAN Huan^3,^***(

)

1 School of Chemical Engineering and Technology, Tianjin University, Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin 300072, China
2 Tianjin R&D Center for Petrochemical Technology, Tianjin University, Tianjin　300072, China
3 Tianjin Animal Science and Veterinary Research Institute, Tianjin　300381, China

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摘要：

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。

关键词： 酿酒酵母; UPRE-lacZ; UPR响应; 抑制物; 分泌表达

Abstract:

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.

Key words: Yeast UPRE-lacZ UPR response Inhibitor tolerance Secretory expression

收稿日期: 2020-06-22 出版日期: 2020-11-10

ZTFLH:

Q291

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

通讯作者: 邹少兰,范寰 E-mail: slzhou@tju.edu.cn;fanhuan1971@163.com

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引用本文:

章小毛,郭敬涵,洪解放,陆海燕,丁娟娟,邹少兰,范寰. UPRE-lac Z为报告基因评价酵母UPR响应初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(10): 1-9.

ZHANG Xiao-mao,GUO Jing-han,HONG Jie-fang,LU Hai-yan,DING Juan-juan,ZOU Shao-lan,FAN Huan. Evaluation of UPR Response in Yeast by Using UPRE-lac Z as a Reporter Gene. China Biotechnology, 2020, 40(10): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006036 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I10/1

表1 实验所用菌株和质粒

表2 实验所用引物

图1 PCR片段琼脂糖凝胶电泳图谱和供体DNA片段示意图

图2 平板菌落显色(左W303-1A宿主,右An-a宿主)

图3 PCR鉴定产物琼脂糖凝胶电泳(左W303-1A宿主,右An-a宿主)

图4 四个菌株的生长曲线

图5 不同添加物培养基中限氧培养4 h时的OD600值和β-半乳糖苷酶酶活

图6 菌株WZ和AZ YPC中生长24 h时的酶活

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