全局转录机制工程法筛选丁醇耐受大肠杆菌及性质

Screening of Butanol-Tolerant Escherichia coli Strain by Global Transcription Machinery Engineering and Its Characterization

作者旨在采用一种快速有效的方法(全局转录机制工程方法)筛选一株具有较高丁醇耐受性的大肠杆菌。通过对全局转录因子σ^(70)进行随机突变,构建rpoD(编码σ^(70)因子)突变库。并采用高通量筛选的方法筛选获得丁醇耐受突变株E. coli JM109/pHACM^(B8),并对该突变株的性质进行了研究。结果表明:丁醇耐受突变株B8能够耐受体积分数2%丁醇,且对其他有机溶剂也具有较高的耐受性,如体积分数3%异丁醇、体积分数8%乙醇、体积分数4%环己烷和体积分数0.3%甲苯。为进一步阐明了B8菌株丁醇耐受机制,分别对B8菌株和对照菌株E. coli JM109/p HACM^(WT)的生理特性进行了研究。结果显示突变株B8胞内的丁醇含量较对照菌株低55%;且在酸性环境下比对照菌株生长更好;此外Mg^(2+)有助于提高菌株B8的丁醇耐受性。本研究为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。

In this study,we employed a fast and efficient approach,Global Transcription Machinery Engineering(gTME),to improve the butanol tolerance of Escherichia coli. The results show that one mutant E. coli JM109/pHACMB8 harboring rpoD mutant B8 could tolerate 2%(v/v) butanol. Further studies showed that the mutant^B8 also exhibited high tolerance to other organic solvents such as 3%(v/v) isopropanol,8%(v/v) ethanol,4%(v/v) cyclohexane and 0.3%(v/v) toluene. To further reveal the tolerance mechanism of the mutant,we investigated the physiological characteristics of both E. coli JM109/pHACM^WT and the mutant B8. The results show that the intracellular butanol concentration of mutant B8 was 55% lower and has improved acid tolerance compared with WT. In addition,Mg^2+ was proved to have a beneficial effect on the butanol tolerance of B8 mutant. Our study provides experimental data and theoretical basis for constructing OST bacteria for industrial applications.