MRP8过表达促进重组酿酒酵母外切纤维素酶生产

doi:10.13560/j.cnki.biotech.bull.1985.2018-0380

摘要/Abstract

摘要： 增强酿酒酵母纤维素酶分泌能力,为提高利用联合生物加工生产纤维素乙醇的效率提供基础。采用CRISPR/Cas9基因组编辑技术,在分泌表达外切纤维素酶CBH1的酿酒酵母Y294中过表达线粒体核糖体蛋白基因MRP8。与对照菌株相比,过表达MRP8重组酵母的胞外CBH1酶活提高了约80%。实时定量PCR结果分析表明,在MRP8过表达突变体中,CBH1转录水平高于对照菌株,但是与蛋白折叠和分泌相关的关键基因转录水平没有明显变化。在刚果红平板和含有衣霉素或二硫苏糖醇的平板上生长没有受到影响。胞内ATP含量和活性氧积累未发现显著差别。本研究表明MRP8过表达促进外切纤维素酶的生产。

关键词: 酿酒酵母, 异源蛋白表达, 外切纤维素酶, 线粒体核糖体蛋白基因MRP8, 蛋白合成

Abstract: Cellulase production and secretion using budding yeast Saccharomyces cerevisiae as a host is one of the strategies for the production of cellulosic ethanol by consolidated bioprocessing（CBP）. However,the ability of cellulase secretion by S. cerevisiae still needs to be improved. Therefore,the aim of this study is to improve cellulase production by the recombinant yeast. It was reported that elevation of genes involved in oxidative stress tolerance improved cellulase production,we thus investigate the effect of overexpression of MRP8,which encodes the mitochondrial ribosomal protein in the recombinant yeast producing CBH1. MRP8 was overexpressed through CRISPR/Cas9 genome editing in the recombinant S. cerevisiae strain producing CBH1. The underlying mechanisms for improved CBH1 production were explored. Results showed that omparing with the control strain,the CBH1 activity was improved by 80% in the MRP8 overexpression strain,and no significant effect on growth ability was observed by MRP8 overexpression. Real-time quantitative PCR analysis showed that the transcription level of CBH1 was increased in the recombinant strain overexpressing MRP8,whereas,the transcription levels of key genes related to protein folding and secretion exhibited no change. No difference on cell growth in the presence of Congo red,Tunicamycin（TM）or Dithiothreitol（DTT）was observed. In addition,no significant difference in ATP content and reactive oxygen species（ROS）accumulation was detected. These results indicate that overexpression of MRP8 enhanced production of CBH1.

Key words: Saccharomyces cerevisiae, heterologous protein expression, cellobiohydrolase 1（CBH1）, mitochondrial ribosomal protein encoding gene MRP8, protein synthesis

万青青,李洁,曾钰,张明明,赵心清,白凤武. MRP8过表达促进重组酿酒酵母外切纤维素酶生产[J]. 生物技术通报, 2018, 34(5): 94-100.

WAN Qing-qing, LI Jie, ZENG Yu, ZHANG Ming-ming, ZHAO Xin-qing, BAI Feng-wu. Enhanced Production of Cellobiohydrolase in the Recombinant Saccharomyces cerevisiae by MRP8 Overexpression[J]. Biotechnology Bulletin, 2018, 34(5): 94-100.

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