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Escherichia coli is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized E. coli strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene rraA , SuptoxR can suppress the high toxicity, which is frequently observed during the MP-overexpression process, and, at the same time, enhance significantly the cellular accumulation of membrane-incorporated and properly folded recombinant MP. The combination of these two beneficial effects results in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic MPs. Here, we engineered second-generation SuptoxR strains with further improved properties, so that they can achieve even higher levels of recombinant MP production. We searched for naturally occurring RraA variants with similar or improved MP toxicity-suppressing and production-promoting effects to that of the native E. coli RraA of the original SuptoxR strain. We found that the RraA proteins from Proteus mirabilis and Providencia stuartii can be even more potent enhancers of MP productivity than the E. coli RraA. By exploiting these two newly identified RraAs, we constructed two second-generation SuptoxR strains, termed SuptoxR2.1 and SuptoxR2.2, whose MP-production capabilities often surpass those of the original SuptoxR significantly. SuptoxR2.1 and SuptoxR2.2 are expected to become widely useful expression hosts for recombinant MP production in bacteria.

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.

Background: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome., Results: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences., Conclusions: This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.