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.
作者
曾钰
熊亮
李洁
DENHAAN Riaan
赵心清
白凤武
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)
出处
《菌物学报》
CAS
CSCD
北大核心
2018年第11期1479-1488,共10页
Mycosystema
基金
国家自然科学基金(31461143029
51561145014)~~