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2025年4月4日 星期五
农业生物技术学报  2020, Vol. 28 Issue (10): 1849-1861    DOI: 10.3969/j.issn.1674-7968.2020.10.013
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
不同启动子及锚定蛋白对酿酒酵母表面展示β-葡萄糖苷酶活性的影响
周红1,*, 张阳1,2,*, 宋育阳1, 李莹1, 杜青1, 张会宁3, 刘延琳1**
1 西北农林科技大学 葡萄酒学院,杨凌 712100;
2 茅台学院,仁怀 564500;
3 扬州中瑞酒店职业学院,扬州 225002
Effects of Different Promoters and Anchoring Proteins on β-glucosidase Activity Displayed on the Surface of Saccharomyces cerevisiae
ZHOU Hong1,*, ZHANG Yang1,2,*, SONG Yu-Yang1, LI Ying1, DU Qing1, ZHANG Hui-Ning3, LIU Yan-Lin1,**
1 College of Enology, Northwest A & F University, Yangling 712100, China;
2 Moutai Institute, Renhuai 564500, China;
3 Yangzhou Hospitality Institute, Yangzhou 225002, China
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摘要 β-葡萄糖苷酶(β-glucosidase, BGL)常作为限速酶在生物能源以及食品领域起着重要作用,本研究通过酿酒酵母(Saccharomyces cerevisiae)表面展示系统中的启动子和锚定蛋白的筛选来提高BGL的酶活。从高拷贝质粒pYES2/CT/α-factor出发,GFP作为报告蛋白,来源于黑曲霉(Aspergillus niger)的BGL作为目标蛋白,构建3种锚定蛋白(α-凝集素蛋白(mating agglutinin protein, Sag1p), 抑制指数缺陷蛋白(suppression of exponential defect protein, Sed1p)和细胞壁蛋白2 (cell wall protein 2, Cwp2p))表面展示GFP的重组酵母和两种启动子(抑制指数缺陷蛋白的启动子(promoter of suppression of exponential defect protein, SED1)和3-磷酸甘油脱氢酶的启动子(promoter of glycerol 3-phosphate dehydrogenase, GPD)和GPD))与3种锚定蛋白(Sag1p, Sed1p和Cwp2p)组合配对表面展示BGL的重组酵母,探究不同启动子和锚定蛋白对酿酒酵母表面展示BGL活性的影响。利用激光共聚焦显微镜观察绿色荧光蛋白发现成功搭建酿酒酵母表面展示平台;成功构建了6株由两种启动子SED1和GPD以及3种锚定蛋白Sag1p、Sed1p和Cwp2p组合搭配的表面展示BGL的重组酵母。结果表明,在6株重组酵母中,GPD启动子比SED1启动子的效果更好;对两种启动子而言,在3种锚定蛋白(Sag1p, Sed1p, Cwp2p)中,Sag1p展示的酶活最高;GPD启动子与Sag1p锚定蛋白组合表面展示BGL的重组酵母PBy-GBSa的酶活最高,最高酶活为(18.29±1.05) U/g。结果表明不同启动子及锚定蛋白对酿酒酵母表面展示BGL有重要影响。本研究为以后酿酒酵母更加高效稳定地表面展示BGL提供理论指导,为实现BGL全细胞催化剂工业化应用提供理论基础。
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周红
张阳
宋育阳
李莹
杜青
张会宁
刘延琳
关键词 酵母表面展示锚定蛋白启动子β-葡萄糖苷酶    
Abstract:β-glucosidase plays a vital role as a rate-limiting enzyme in the field of bioenergy and food industries. In this study, different promoters and anchor proteins were screened in the surface display system of Saccharomyces cerevisiae to explore the effects of different promoters and anchor proteins on the BGL activity from Aspergillus niger. pYES2/CT/α-factor was used as the starting plasmid, green fluorescent protein (GFP) as the reporter protein and mating agglutinin protein (Sag1p), suppression of exponential defect protein (Sed1p) and cell wall protein 2 (Cwp2p) as anchors protein, to construct 3 different surface display plasmids of anchor protein GAL1-eGFP-Sag1/Sed1/Cwp2 and yeast surface display system. The original promoter GAL1 of plasmid pYES2/CT/α-factor was replaced with constitutive promoters promoter of glycerol 3-phos‐phate dehydrogenase (GPD) and promoter of suppression of exponential defect protein (SED1) by In-Fusion technology. Plasmids pYES2-GPD and pYES2-SED were constructed and digested , then they were separately ligated with the digested GAL1-eGFP-Sag1/Sed1/Cwp2 constructed above to construct 6 recombinant plasmids GAP/SED1-eGFP-Sag1/Sed1/Cwp2. Finally, the eGFP on the plasmids GPD/SED1-eGFP-Sag1/Sed1/Cwp2 were replaced with the target protein gene bgl1 derived from A. niger and 6 recombinant yeasts, PBy-GBSa, PBy-GBSe, PBy-GBCw, PBy-SBSa, PBy-SBSe, PBy-SBCw, were successfully constructed to explore the effects of different promoters (SED1 and GPD) and anchoring proteins (Sag1p, Sed1p and Cwp2p) on BGL activity. Green fluorescent protein was observed by laser confocal microscope and was found to be located on the cell surface of S. cerevisiae, revealing that the surface display platform of S. cerevisiae was successfully established. The activities of BGL displayed on the surface of 6 recombinant yeasts were compared with each other, which showed that the GPD promoter performed better than the SED1 promoter; no matter what kind of promoter, Sag1p showed the highest enzyme activity among the 3 anchor proteins (Sag1p, Sed1p, Cwp2p); When GPD was used as the promoter, the differences in enzyme activity between different anchor proteins were more obvious. The BGL recombinant strain PBy-GBSa was able to display BGL more efficiently, and the enzyme activity reached a maximum of (18.29±1.05) U/g when cultured for 24 h. The types of promoters and anchoring proteins had important effects on the surface display of BGL in S. cerevisiae, which would provide a theoretical basis for the more efficient and stable surface display of BGL in S. cerevisiae and the industrial application of BGL whole-cell catalysts.
Key wordsYeast surface display    Anchor protein    Promoter    β-glucosidase
收稿日期: 2020-02-22     
ZTFLH:  S182  
  S188  
  Q78  
基金资助:国家重点研发计划项目(2019YFD1002500); 国家自然科学基金(31501463); 农业农村部葡萄酒加工重点实验室开放课题(KLVE201702); 国家葡萄产业技术体系(CARS-29-jg-03)
通讯作者: * yanlinliu@nwsuaf.edu.cn   
作者简介: *同等贡献作者
引用本文:   
周红, 张阳, 宋育阳, 李莹, 杜青, 张会宁, 刘延琳. 不同启动子及锚定蛋白对酿酒酵母表面展示β-葡萄糖苷酶活性的影响[J]. 农业生物技术学报, 2020, 28(10): 1849-1861.
ZHOU Hong, ZHANG Yang, SONG Yu-Yang, LI Ying, DU Qing, ZHANG Hui-Ning, LIU Yan-Lin. Effects of Different Promoters and Anchoring Proteins on β-glucosidase Activity Displayed on the Surface of Saccharomyces cerevisiae. 农业生物技术学报, 2020, 28(10): 1849-1861.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.10.013     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I10/1849
 
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