Second-Generation SuptoxR Strains for High-Level Recombinant Membrane Protein Production.

is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene , 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 RraA of the original SuptoxR strain. We found that the RraA proteins from and can be even more potent enhancers of MP productivity than the 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.