Gialama Dimitra, Kostelidou Kalliopi, Michou Myrsini, Delivoria Dafni Chrysanthi, Kolisis Fragiskos N, Skretas Georgios
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation , Athens 11635, Greece.
Laboratory of Biotechnology, School of Chemical Engineering, National Technical University of Athens , Athens 15780, Greece.
ACS Synth Biol. 2017 Feb 17;6(2):284-300. doi: 10.1021/acssynbio.6b00174. Epub 2016 Nov 17.
Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein production. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, respectively, accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of commercial strains frequently utilized for recombinant membrane protein production purposes.
膜蛋白在所有生物中执行关键的细胞功能，并且是药物研发的主要靶点。大肠杆菌一直是用于膜蛋白生化/结构研究的最常用的过表达宿主。然而，重组膜蛋白的细菌生产通常受到细胞积累不佳和对宿主的严重毒性的阻碍，这导致最终生物量较低和体积产量微小。在这项工作中，我们旨在重新调整大肠杆菌的蛋白质生产机制，以承受膜蛋白过表达引起的毒性，从而产生具有实现高水平膜蛋白生产能力的工程菌株。为了实现这一目标，我们寻找其共表达可以抑制膜蛋白诱导的毒性的细菌基因，并鉴定出两种高效效应物：膜结合的DnaK共伴侣DjlA和大肠杆菌核糖核酸酶E的mRNA降解活性抑制剂RraA。分别共表达djlA或rraA的大肠杆菌菌株，分别称为SuptoxD和SuptoxR，积累了明显更高水平的最终生物量，并且为多种原核和真核重组膜蛋白产生了显著提高的产量。在所有测试的情况下，SuptoxD、SuptoxR或两者都优于常用于重组膜蛋白生产目的的商业菌株的能力。
