Extracellular peptide production in Escherichia coli by inducible downregulation of lipoprotein Lpp via MicL sRNA.

Abstract

Despite its many benefits, Escherichia coli only poorly secretes recombinant proteins and peptides into the medium. This complicates downstream processing and notably contributes to the production costs of biopharmaceuticals. The permeability of production strains can be increased by deletion of the lpp gene, coding for Braun's lipoprotein Lpp. Consequently, the outer membrane (OM) is destabilized, and periplasmic recombinant proteins/peptides can leak out of the cell into the cultivation medium. However, we observed poor process performance during C-limited fed-batch cultivations in bioreactors when production strains with lpp knockout were cultivated. In this study, we developed an inducible system for in-process Lpp downregulation (knockdown) in E. coli with the goal to facilitate the release of the periplasmic recombinant fusion peptide CASPON-SST into the cultivation medium. By plasmid-based overexpression of MicL sRNA, we were able to efficiently inhibit Lpp synthesis and increase the OM permeability of our production strains. With this approach, we were able to achieve the secretion of 80-100% of all peptide and increased production capacities. The system was further optimized by utilizing different promoter systems to induce peptide and MicL expression separately in order to coordinate them. We report here for the first time the extracellular production of a recombinant peptide by inducible downregulation of Lpp via MicL sRNA during C-limited fed-batch cultivations. By utilizing a flexible system for Lpp knockdown, potential drawbacks of lpp knockout can be counteracted, thus making our approach a valuable tool for the in-process adaptation of OM permeability in production hosts. KEY POINTS: • Lpp can be downregulated on mRNA level by MicL sRNA overexpressed during fed-batch. • Recombinant peptides can leak out of the periplasm when Lpp is downregulated. • Leakiness and outer membrane permeability must be distinguished.