Functional Requirements for DjIA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR

In previous work, we have generated the engineered Escherichia coli strains SuptoxD and SuptoxR, which upon co-expression of the effector genes djIA or rraA, respectively, are capable of suppressing the cytotoxicity caused by membrane protein (MP) overexpression and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Here, we investigated the functional requirements for DnaJ-/ike protein A (DjIA)- and regulator of ribonuclease activity A (RraA)-mediated enhancement of recombinant MP production in these strains and show that: (i) DjIA and RraA act independently, that is, the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjIA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjIA in SuptoxD involve the action of the molecular chaperone DnaK but do not rely on the activation of the regulation of capsular synthesis response, a well-established consequence of djIA overexpression; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E, but not that of its paralogous ribonuclease RNase G; and (v) DjIA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production. These observations provide important clues about the molecular requirements for suppressed toxicity and enhanced MP accumulation in SuptoxD/SuptoxR and will guide future studies aiming to decipher the exact mechanism of DjIA- and RraA-mediated enhancement of recombinant MP production in these strains. (C) 2017 Elsevier Ltd. All rights reserved.