耐辐射异常球菌irrE表达与简单节杆菌有机溶剂耐受性的关系和基于DNA损伤修复的作用机制研究

The non-line relationship between the gene expression level and the host target phenotype is the key problem, which affects the accurate assessment of gene function and the achievement on the desired phenotype. In this project, a series of recombinant strains with different irrE expression levels within a wide range are obtained by the random mutagenesis of a constitutive promoter for revealing the correlation of irrE gene expression level with the organic-solvent tolerance of Arthrobacter simplex. The optimal expression level is identified and the production performance of the strain with the best organic-solvent tolerance is evaluated. The stains with high expression level, optimal expression level and low expression level are selected as the model to explore the effects of gene expression level on the cell physiological and metabolic properties. The gene expression level difference is further explained by analyzing the mutation site of the promoter with sequence technology and by determining the gene copy number and transcription level with RT-PCR. At the same time, their cell survival and DNA repair process under different solvent stress conditions are investigated. The functional mechanism of IrrE on the damaged DNA repair in Arthrobacter simplex upon organic solvent stress is elucidated by the quantification of the relevant metabolic substance and the analysis of two key proteins invloved in the DNA repair and replication process, RecA and UvrD/REP (identified in our previous experiment) at expression level and transcriptional level. Our results will lay the theoretical foundation and provide technical support for the desired phenotype.

异源基因的表达水平与宿主生物目标表型之间的非线性关系是影响基因功能准确评价和理想目标表型获得的关键问题。项目通过组成型启动子的随机突变构建irrE表达水平分布广泛且差异明显的系列菌株，揭示irrE表达与简单节杆菌有机溶剂耐性之间的关系，确定最适表达水平，对优良菌株的生产性能进行评价。以高表达、最适表达和低表达菌株为对象，考察不同表达水平对菌株生理代谢性能的影响，利用测序和实时荧光定量PCR从启动子突变位点、基因拷贝数和转录水平三方面分析表达水平产生差异的原因,测定不同有机溶剂胁迫条件下细胞的存活和DNA损伤修复情况，通过检测相关的代谢物质含量，分析参与DNA修复和复制过程的两个蛋白RecA和UvrD/REP（课题组前期鉴定的显著差异蛋白）表达水平和基因转录水平的变化，阐述IrrE对简单节杆菌有机溶剂胁迫下DNA损伤修复过程的作用机制。其成果为理想目标表型的获得提供理论基础和技术支持。

简单节杆菌是工业上普遍使用的甾体C1,2脱氢反应菌株，转化体系中多采用添加有机溶剂的方法（一般是乙醇，浓度为4%）促进疏水性底物的溶解。但高浓度有机溶剂对菌体的毒害作用限制了其使用剂量，进而制约底物投料浓度和反应效率。本项目将来源于极端微生物-耐辐射异常球菌中的全局转录因子IrrE导入简单节杆菌，结果发现，IrrE表达对菌株无压力条件下的生长特性、代谢能力、催化酶活力无明显影响，但却显著增强了菌株的有机溶剂耐受性，进而提高其在高浓度有机溶剂和底物转化体系中的生产效率；通过IrrE在革兰氏阴性菌—大肠杆菌和革兰氏阳性菌—球形节杆菌中的表达及其菌株的压力耐受性能分析，确定了irrE基因在不同物种间水平转移的可行性；通过相关代谢物（如活性氧簇水平、甘油和海藻糖、ATP）、关键酶酶活和相关基因转录水平的分析，阐明IrrE表达提高简单节杆菌有机溶剂耐受性能的调控机制主要包括：缓解胞内氧化应激响应、提高胞内相容性溶质含量积累、提高胞内能量水平、增强菌株DNA损伤修复能力、上调热激蛋白表达水平和调控胞内其他全局转录因子的表达水平（如上调与耐受性能密切相关的rpoD表达水平）；通过多轮易错PCR技术构建启动子突变文库，筛选得到IrrE差异表达菌株，通过分析菌株在适当压力添加生长体系和高浓度压力冲击体系下的生长和存活评价其有机溶剂耐受性能；确定IrrE不同表达水平对菌株有机溶剂耐受性能关键途径的影响，结果发现，简单节杆菌中IrrE表达水平越高，菌株内重组酶RecA表达水平和氧化应激响应相关酶的活力越大，菌株DNA损伤修复能力和清除胞内ROS的能力越强，这是IrrE高表达菌株表现出更高有机溶剂耐受性能的重要原因；最后对获得的高有机溶耐性工程菌株的生产能力进行评价，结果表明，该菌株在底物CA浓度35 g/L和乙醇浓度为10%的转化体系中转化48 h后，底物转化率和产物PA生成量均达到最大，分别为60.9%和为21.1 g/L，而对照菌株无明显的转化能力。项目成果对于补充和完善irrE基因在逆境条件下对宿主生物的总体调控机制具有重要的科学意义，也为具有良好工业应用属性的高效甾体C1,2脱氢反应菌株的构建提供新策略和新方法。