Signal Peptide and Chaperone Engineering for Secretion and Excretion in Escherichia coli

Abstract

Secretory proteins play critical role in cell survival and pathogenicity and make up approximately 30% of all polypeptides synthesized by gram-negative bacteria. They may be found in a soluble form in the periplasm (the interstitial space between inner and outer membranes), integrated within the outer membrane or excreted outside of the cell in a vesicular or globular form. In all cases, they must first translocate across the inner membrane. A substantial fraction of secretory proteins do so by making use of a cleavable N-terminal extension called signal sequence (or peptide) that is recognized by a dedicated translocation system known as the Sec pathway. Typical Sec-dependent signal sequences have a tripartite structure with a central hydrophobic core and are about 20 residues in length. However, certain secretory proteins implicated in virulence employ Sec-dependent signal peptides that are over 40 residues long. To determine if repetition of canonical, Sec-dependent signal peptides would benefit secretory protein production, we fused the signal sequences of E. carotovora PelB and E. coli OmpA to one another to produce synthetic PelB-OmpA and OmpA-PelB leader peptides. Using periplasmic maltose binding protein (MBP) and outer membrane protein A (OmpA) as model systems, we found that dual signal peptides support Sec-dependent protein translocation and are preferentially cleaved at the signal peptidase I site vicinal to the mature protein. In the case of native OmpA, dual signal peptides increased the requirement for the molecular chaperone Trigger Factor but reduced the accumulation of misfolded precursor and mature species and delayed the acquisition of PBAD promoter mutations that restore cell growth by shutting down recombinant protein synthesis. On the other hand, dual signal sequences did not improve the incorporation of an OmpA1-183-mCherry fusion protein within extracellular outer membrane vesicles (OMVs), as highest yields were obtained with the PelB signal peptide. However, co-expression of periplasmic chaperones (primarily SurA) and components of the outer membrane protein assembly machinery (primarily BamA) improve OMV production by nearly twofold. Overall, our results highlight the potential and limitations of signal sequence and chaperone engineering strategies for the production of secretory proteins and OMVs in E. coli.