rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

Functions of rpoS gene in response to environmental stress by Enterobacter sp. CGMCC 5087

摘要:

【背景】苯乙醇(2-Phenylethanol，2-PE)是一种具有玫瑰香气味的高级香料添加剂，被广泛应用于香水、化妆品、食品和医药等领域。目前，利用工程菌合成苯乙醇有很好的应用前景。我们分离到一株肠杆菌(Enterobacter sp.) CGMCC 5087，其可以通过苯丙酮酸途径合成2-PE。然而该菌的生长受到不同环境因素导致的胁迫，进而影响苯乙醇的产量。RpoS作为一种稳定期σ因子和压力应答过程中的主要调节因子，在细菌抗环境胁迫生长中起重要作用。【目的】阐明肠杆菌CGMCC 5087中rpoS基因在多种环境胁迫中的作用，掌握该菌在不同环境胁迫下的生物学特性。【方法】使用CRISPR基因编辑技术敲除rpoS基因，通过质粒表达系统构建互补菌株，检测rpoS基因缺失株ΔrpoS与野生型WT菌株和互补菌株ΔrpoS(rpoS)在高渗透压、高温、低pH和氧化应激环境下的生长情况，并进行统计学分析。【结果】rpoS基因的缺失显著降低了肠杆菌CGMCC 5087的生长。在5% NaCl和pH 5.0胁迫条件下，rpoS基因的缺失导致肠杆菌CGMCC 5087的耐受性显著降低。在42 °C高温条件下，rpoS基因的缺失导致肠杆菌CGMCC 5087在对数期的耐受性显著降低，而在衰退期的耐受性增强。1 mmol/L H2O2氧化胁迫条件下，rpoS基因的缺失导致肠杆菌CGMCC 5087的延滞期延长，而进入稳定期后rpoS基因突变株耐受性较野生型菌株明显增强。【结论】在肠杆菌CGMCC 5087中，RpoS在抵抗多种环境压力中均具有重要作用，而且在菌株不同的生长时期对于环境胁迫的应答也有所不同，为进一步了解肠杆菌CGMCC 5087的生物学特性、掌握RpoS在肠杆菌CGMCC 5087合成苯乙醇过程中的作用机制提供基础。

Abstract:

[Background] 2-phenylethanol is a high-grade flavoring additive with rosy scent, and widely used in perfume, cosmetics, food and medicine. At present, the synthesis of 2-phenylethanol by engineering bacteria has a good prospect. We isolated an Enterobacter sp. CGMCC 5087 that can synthesize 2-phenylethanol through phenylpyruvate pathway. However, the growth of the bacterium and 2-phenylethanol yield are affected by environmental stress. As a stationary phase Sigma factor and a major regulator in response to stress, RpoS plays an important role in resistant to environmental stress. [Objective] In order to elucidate the role of rpoS gene in Enterobacter sp. CGMCC 5087 under various environmental stresses. [Methods] Using CRISPR gene editing technology to knockout the rpoS gene, and the complementary strain was constructed by introducing the plasmid containing rpoS gene into ΔrpoS strain. The growth of rpoS gene-deficient strain ΔrpoS, the wild-type strain and complementary strain ΔrpoS(rpoS) under high osmotic pressure, high temperature, low pH and oxidative stress environments was detected and statistically analyzed. [Results] Deletion of rpoS gene significantly reduced the growth of Enterobacter sp. CGMCC 5087 strain. Under the stress of 5% NaCl and pH 5.0, deletion of rpoS gene significantly reduced the tolerance of Enterobacter sp. CGMCC 5087. At 42 °C, the deletion of rpoS gene resulted in a significant decrease in the tolerance of Enterobacter sp. CGMCC 5087 in logarithmic phase, and increased during the decline phase. Under the condition of 1 mmol/L H2O2 oxidation stress, the deletion of rpoS gene led to the prolongation of the delay period of Enterobacter sp. CGMCC 5087, and the tolerance of the mutant strain ΔrpoS was significantly higher than that of the wild type. [Conclusion] In Enterobacter sp. CGMCC 5087, RpoS plays an important role in resisting to various environmental stresses, and the response to environmental stresses varies with the different growth periods of bacteria. The study provides a basis for further understanding of the biological characteristics of Enterobacter sp. CGMCC 5087 and the mechanism of RpoS in the synthesis of 2-phenylethanol of Enterobacter sp. CGMCC 5087.