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MHF组蛋白折叠复合体组分编码基因MHF1过表达对重组酿酒酵母纤维素酶生产的影响
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国家自然科学基金(31461143029,51561145014)


Effects of overexpression of the MHF histone fold complex component encoding gene MHF1 on production of cellulase by the recombinant Saccharomyces cerevisiae
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    摘要:

    【背景】重组酿酒酵母可用于生产多种药用蛋白和工业酶等外源蛋白,但蛋白分泌水平低是限制其异源蛋白高效生产的重要因素。异源蛋白表达和分泌过程可能会对宿主细胞产生多种胁迫,因此,研究胁迫响应相关基因对重组酵母异源蛋白生产的影响具有重要意义。Mhf1p是MHF组蛋白折叠复合体的组分之一,与DNA损伤修复及维持基因组稳定性有关,但其对异源蛋白生产的作用尚不清楚。【目的】研究MHF1过表达对重组酿酒酵母蛋白生产的影响。【方法】在分泌表达纤维素酶的重组酿酒酵母菌株中利用基于CRISPR-Cas9的基因组编辑技术整合过表达MHF1,分析其对产酶的影响,并探讨影响产酶的分子机理。【结果】与出发菌株相比,过表达MHF1菌株的外切纤维素酶CBH酶活性提高了38%。对过表达MHF1的CBH生产菌株中蛋白合成和分泌途径相关基因转录水平进行检测,发现与对照菌株相比,CBH1基因和与分泌相关的SEC22、ERV29等基因在不同时间点呈现不同程度显著上调。【结论】MHF1过表达可促进酿酒酵母异源外切纤维素酶的生产,并影响外源酶基因和分泌途径基因的表达,可能通过对多基因的协同表达影响促进产酶。

    Abstract:

    [Background] Saccharomyces cerevisiae is widely used to produce valuable proteins such as pharmaceutical proteins and industrial enzymes, but the low protein production and secretion level are challenging for efficient production of heterologous proteins. Synthesis of heterologous proteins and their secretion may produce a variety of stresses to host cells, thereby inhibit the production efficiency. Therefore, studying the effects of stress responsive genes on heterologous protein production is of great significance. Mhf1p is one of the components of MHF histone folding complex, and involved in repairing damaged DNA and maintenance of genome stability, but its role in the production of heterologous proteins remains unclear. [Objective] To study whether MHF1 overexpression can promote protein production in the recombinant S. cerevisiae. [Methods] MHF1 was overexpressed by chromosomal integration in the recombinant S. cerevisiae strains producing different cellulases employing CRISPR-Cas9 based genome editing, and cellulases secretion was compared with that of the control strain. Furthermore, the underlying molecular mechanisms were explored. [Results] Compared with that of the control strain, cellobiohydrolase (CBH) activity in the MHF1 overexpressing strain was increased by 38%. Transcription levels of key genes such as genes related to protein production and secretion of the yeast strains were further detected. Comparing with that of the parent strain, elevated transcription levels of CBH1, as well as key genes related to protein secretion such as SEC22 and ERV29 in different time points were revealed by overexpression of MHF1. [Conclusion] Overexpression of MHF1 promoted the production of a heterologous cellobiohydrolase in S. cerevisiae. Enhanced transcription of CBH1 and genes involved in secretion pathway was revealed, indicating that MHF1 modulates heterologous protein production by synergistic regulation of multiple genes.

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于欣水,曾钰,李洁,张明明,白凤武,赵心清. MHF组蛋白折叠复合体组分编码基因MHF1过表达对重组酿酒酵母纤维素酶生产的影响[J]. 微生物学通报, 2019, 46(1): 75-83

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  • 在线发布日期: 2018-12-28
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