title: Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production.
abstract: 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.
authors: 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.
authors: Dimitra
authors: D
authors: Gialama
authors: Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation , Athens 11635, Greece.
authors: Kalliopi
authors: K
authors: Kostelidou
authors: Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation , Athens 11635, Greece.
authors: Myrsini
authors: M
authors: Michou
authors: 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.
authors: Dafni Chrysanthi
authors: DC
authors: Delivoria
authors: Laboratory of Biotechnology, School of Chemical Engineering, National Technical University of Athens , Athens 15780, Greece.
authors: Fragiskos N
authors: FN
authors: Kolisis
authors: Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation , Athens 11635, Greece.
authors: Georgios
authors: G
authors: Skretas
mesh_terms: D018533:Biomass; D004722:Endoribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: C494634:DjlA protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C477942:RraA protein, E coli; D004722:Endoribonucleases; C022537:ribonuclease E
keywords: DjlA; Escherichia coli; RraA; membrane protein overexpression; toxicity