Improvement of organic solvent tolerance level by directed evolution of AcrAB-TolC efflux pump in Escherichia coli

几种生理和生物化学方法已被应用在努力阐明微生物耐溶剂的机制。近年来，研究表明革兰氏阴性菌对有机溶剂的耐受性与能量依赖性外排有关。编码属于抗性/增殖/细胞分裂（RND）家族的外排泵组分的克隆基因已显示用于维持某些细菌中的溶剂抗性。遗传学证据表明，作为RND家族成员的AcrAB-TolC外排泵参与了大肠杆菌的耐溶剂性。coli细胞。我们表明AcrAB-TolC泵实际上降低了暴露于溶剂的大肠杆菌细胞中的细胞内溶剂浓度。在这项研究中，我们估计是否在大肠杆菌中的有机溶剂水平提高通过使用体外定向进化。采用家族改组和易错PCR技术构建了acrB基因的体外定向进化突变体库。携带突变acrB基因的大肠杆菌菌株的有机溶剂耐受水平降低。该结果表明，构建了外排功能改变的突变体AcrB。我们从AcrAB同源物中寻找比AcrAB-TolC泵活性更高的其他外排泵。结果表明，AcrAB-TalC泵比其他测试的AcrAB同系物显示出更高的活性。

Several physiological and biochemical approaches have been applied in an effort to elucidate the mechanisms of microbial resistance to solvents. In recent years, it has been demonstrated that energy-dependent efflux is involved in organic solvent tolerance in gram-negative bacteria. Cloned genes coding for components of efflux pumps belonging to the resistance/nodulation/cell division (RND) family have been shown to serve to maintain solvent resistance in certain bacteria. Genetic evidence suggests that the AcrAB-TolC efflux pump, a member of the RND family, is involved in solvent resistance of E. coli cells. We showed that the AcrAB-TolC pump actually reduces the intracellular solvent concentration in E.coli cells exposed to a solvent. In this study, we estimated whether the organic solvent level in E.coli improves by using in vitro directed evolution. The family shuffling method and error prone PCR were applied for in vitro directed evolution to construct a mutant library of acrB genes. The organic solvent tolerance levels of E.coli strains harboring mutant acrB genes were lowered. This result shows that mutant AcrB, of which efflux function was changed, was constructed. We searched other efflux pump that has higher activity than that of AcrAB-TolC pumps from AcrAB homologues. As the results, it was shown that AcrAB-TalC pump shows higher activity than other AcrAB homologues tested.