基于膜压力应答途径的大肠杆菌丁醇耐受性

Improving n-butanol tolerance of Escherichia coli based on membrane stress response pathways

摘要:

[目的]筛选丁醇压力下Escherichia coli中参与溶剂压力应答的细胞信号传导途径，并从应答途径出发，提高E.coli丁醇耐受性。[方法]在丁醇压力下，利用RT-PCR分析大肠杆菌内膜压力应答途径中反应调节因子（response regulator，RR）的表达水平，通过Red同源重组以及一步克隆的方法分别构建外膜脂蛋白NlpE和分子伴侣蛋白Spy的敲除菌株E.coli JM109（ΔnlpE）和E.coli JM109（Δspy）及重组菌株E.coli JM109/pQE80L-nlpE和E.coli JM109/pQE80L-spy，并测定其溶剂耐受性和细胞膜疏水性。[结果]0.8%（V/V）丁醇处理10 h后，Cpx和Bae双组分压力应答途径中的cpxR和baeR基因的表达水平分别提高了8.3和3.3倍；分别在含0.6%（V/V）四氢呋喃、0.1%（V/V）甲苯和0.6%（V/V）环己烷的培养基中培养10 h后，重组菌株E.coli JM109/pQE80L-spy和E.coli JM109/pQE80L-nlpE的OD₆₀₀相比对照组（OD₆₀₀增长0.02-0.04）分别增长了0.13-0.17和0.05-0.13，重组菌的溶剂耐受性得到了显著提高。[结论]Cpx和Bae系统参与大肠杆菌丁醇压力应答，分子伴侣蛋白Spy的过表达能够有效提高大肠杆菌对有机溶剂的耐受性，本研究为阐明微生物有机溶剂耐受性机制提供了理论依据。

Abstract:

[Objective] To improve n-butanol tolerance of Escherichia coli by screening and engineering the signal transduction pathways thinvolved in solvent stress response.[Methods] Under butanol stress, expression of response regulator in membrane signal transduction pathways of E. coli was determined and analyzed using RT-PCR. Key regulating components of stress response pathway were deleted and over-expressed in E. coli through red-homologous recombination and one-step clone. Solvent tolerance and membrane hydrophobicity analysis of the deleted and over-expressed strains were conducted against six different organic solvents.[Results] Expression level of cpxR and baeR in two-component stress response pathways Cpx and Bae was increased by 8.3 and 3.3 folds, respectively, after n-butanol (0.8%, V/V) treatment for 10 h. Under the solvent stress of tetrahydrofuran (0.6%, V/V), toluene (0.1%, V/V) and cyclohexane (0.6%, V/V) for 10 h, the OD₆₀₀ of recombinant E. coli JM109/pQE80L-spy and E. coli JM109/pQE80L-nlpE were increased by 0.13-0.17 and 0.05-0.13, respectively, compared with the control (ΔOD₆₀₀ of 0.02-0.04). Organic solvent tolerance of E. coli was improved.[Conclusion] Two component stress response pathways, Bae and Cpx, participate in the response of butanol stress. Overexpression of Spy could effectively improve organic solvent tolerance of E. coli. This study provides theoretical guidance for elucidating the mechanisms of microbial organic solvents tolerance.