破坏细胞壁蛋白CWP2基因提高重组酿酒酵母β-葡萄糖苷酶胞外酶活  

Disrupting cell wall protein encoding gene CWP2 enhances extracellularβ-glucosidase activity by recombinant Saccharomyces cerevisiae

作　　者：李洁 曾钰 张明明 白凤武[1] 赵心清[1] LI Jie;ZENG Yu;ZHANG Ming-Ming;BAI Feng-Wu;ZHAO Xin-Qing(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]微生物代谢国家重点实验室,上海交通大学生命科学技术学院,上海200240

出　　处：《微生物学通报》2020年第3期681-690,共10页Microbiology China

基　　金：国家自然科学基金(31461143029,51561145014)。

摘　　要：【背景】重组酿酒酵母广泛应用于生产工业酶和药用蛋白,但是目前仍旧存在异源蛋白产量低、分泌效率差的问题,限制了生产应用。【目的】提高重组酿酒酵母异源分泌蛋白的能力,构建高效的异源蛋白生产细胞工厂。【方法】采用基于CRISPR/Cas9的基因组编辑技术,以生产β-葡萄糖苷酶的重组酿酒酵母Y294-BGL为出发菌株,构建细胞壁蛋白基因CWP2破坏菌株。【结果】与出发菌株相比,破坏CWP2的破坏菌株在发酵96 h时胞外β-葡萄糖苷酶酶活可提高53%,胞内酶活提高了208%。此外,破坏菌生长未受到影响,对弱酸等环境胁迫的耐性没有下降,未造成过多内质网胁迫。进一步检测发现,破坏菌株胞内活性氧水平下降,同时蛋白胞内运输和分泌途径相关的关键基因表达转录及多个细胞壁生物合成相关基因表达下降。【结论】破坏细胞壁蛋白基因CWP2能够提高异源蛋白β-葡萄糖苷酶的胞外酶活,可作为促进酿酒酵母生产异源蛋白的靶点基因。[Background]Saccharomyces cerevisiae is widely used to produce industrial enzymes and pharmaceutical proteins.However,low protein production level and poor secretion efficiency are the major bottlenecks for its industrial applications.[Objective]To improve protein production by the recombinant yeast strains,and provide basis for development of robust yeast cell factory for heterologous protein secretion.[Methods]The cell wall protein encoding gene CWP2 was disrupted through CRISPR/Cas9-based genome editing in the recombinant strain S.cerevisiae Y294-BGL,which can secretβ-glucosidase.[Results]At 96 h of fermentation,the extracellular activity ofβ-glucosidase in the mutant Bcwp2△was improved by 53%,and intracellular activity was improved by 208%.No negative effect on cell growth was observed,and no significant change was observed in the tolerance to acetic acid and ethanol in the mutant yeast strain Bcwp2△.In addition,no difference in growth of the mutant in the presence of the endoplasmic reticulum stress inducers dithiothreitol and tunicamycin was observed.Further studies showed that less reactive oxygen species was accumulated inside the cells of the mutant.Disruption of CWP2 decreased transcription of genes associated with protein trafficking and secretion,as well as cell wall biosynthesis genes.[Conclusion]Disruption of the cell wall protein encoding gene CWP2 promotes extracellularβ-glucosidase activity,and can be employed as a target in metabolic engineering of yeast protein production.

关 键 词：纤维素酶 酿酒酵母 Β-葡萄糖苷酶 CWP2 异源蛋白表达 

分 类 号：Q55[生物学—生物化学]