Ni Ye, Reye John, Chen Rachel R
Chemical Engineering Department, Virginia Commonwealth University, Richmond, Virginia 23284, USA.
Biotechnol Bioeng. 2007 Aug 15;97(6):1347-56. doi: 10.1002/bit.21375.
Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.
我们早期对大肠杆菌外膜通透性的研究表明，lpp基因（编码布劳恩脂蛋白）中的插入突变极大地改变了外膜通透性，导致全细胞催化反应显著加速。为了从机制上理解通透性变化的本质，我们对lpp区域进行了测序。结果显示，插入突变体中Lpp未表达，这表明Lpp的缺失而非改变是观察到的通透性变化的原因。这一惊人结果促使我们研究将lpp缺失作为一种通用通透化方法的可能性。我们从具有不同遗传背景的菌株中构建了两个lpp缺失突变体，并研究了lpp缺失对细胞生理学的影响。虽然lpp缺失对细胞生长、碳代谢和脂肪酸组成没有显著影响，但它增强了各种小分子的通透性，这与插入突变体的结果一致。这种表型在广泛的生物技术应用中很有用。我们在此展示了该突变体在有机磷酸水解和L-肉碱合成中的应用，在这些过程中，通透性是一个限制因素。由于通过外膜的通透性增强，这两个过程在突变体中都得到了显著改善。因此，本研究建立了一种简单且普遍适用的方法来通透化大肠杆菌细胞，且没有明显的不良影响。此外，由于已知革兰氏阴性细菌中存在lpp同源物，我们预计这种方法将适用于其他革兰氏阴性细菌。
