UTH1基因破坏提高重组酿酒酵母分泌β-葡萄糖苷酶  

Disruption of UTH1 improves β-glucosidase secretion by the recombinant Saccharomyces cerevisiae

作　　者：曾钰 熊亮[2] 李洁 DENHAAN Riaan 赵心清[1] 白凤武[1] ZENG Yu;XlONG Liang;LI Jie;DEN HAAN Riaan;ZHAO Xin-Qing;BAI Feng-Wu(School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;School of Life Science and Biotechnology,Dalian University of Technology,Dalian,Liaoning 216024,China;Department of Biotechnology,University of Western Cape,Bellville 7530,South Africa)

机构地区：[1]上海交通大学生命科学技术学院,上海200240 [2]大连理工大学生命科学与技术学院,辽宁大连116024 [3]Department of Biotechnology,University of Western Cape

出　　处：《菌物学报》2018年第11期1479-1488,共10页Mycosystema

基　　金：国家自然科学基金(31461143029;51561145014)~~

摘　　要：异源蛋白质分泌效率低限制了重组酿酒酵母的多种药用蛋白和工业酶生产。挖掘促进蛋白质生物合成和分泌的关键基因,是提高异源蛋白质生产效率的重要手段。酿酒酵母细胞壁完整性影响异源蛋白质分泌,本研究利用基于CRISPR/Cas9的基因组编辑技术,破坏了重组酿酒酵母Y294-BGL1中参与细胞壁合成的未知功能基因UTH1,发现所获得的突变体胞外β-葡萄糖苷酶酶活比出发菌株提高112.9%,而细胞壁完整性下降。对促进产酶的分子机理进行探索,发现突变体产酶条件下与细胞壁完整性相关的关键基因和与蛋白质分泌途径相关的基因转录出现明显差异,提示UTH1基因破坏不仅影响细胞壁完整性关键基因的表达,也影响蛋白质分泌途径。本文的研究结果有助于深入理解UTH1的基因功能,并为构建异源蛋白质高分泌酵母菌株提供了借鉴。Low protein secretion efficiency limited the applications of Saccharomyces cerevisiae for production of various pharmaceutical proteins and industrial enzymes. Mining new key genes to promote protein biosynthesis and secretion enhances the production efficiency of heterologous proteins. It has been reported that cell wall integrity affects the secretion of heterologous proteins. In this study, UTH1, which encoded a protein with unknown function and was found to be involved in cell wall synthesis, was disrupted via the CRISPR/Cas9-based genome editing in the β-glucosidase（BGL1） strain Y294-BGL, and the enzyme activity of heterologous BGL1 in the UTH1-disruptant increased by 112.9%, while cell wall integrity decreased. Further studies showed that the transcription levels of key genes related to cell wall integr ity and some key genes involved in protein secretion changed significantly in the UTH1-disruptant, indicating that disruption of UTH1 affected not only genes related to cell wall integrity but also genes involved in protein secretion. The results in this study may promote the understanding of the function of UTH1 in S. cerevisiae, and benefit further construction of robust yeast strains to produce heterologous proteins.

关 键 词：纤维素酶 酿酒酵母 Β-葡萄糖苷酶 UTH1基因 细胞壁完整性 

分 类 号：Q78[生物学—分子生物学]