Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress

In Escherichia coli many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing target gene expression intensity with the Sec-translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec-translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm and an inefficient energy metabolism result in poor growth and low protein production yields. Harmonizing target gene expression intensity with the Sec-translocon capacity results in normal growth, enhanced protein production yields and, surprisingly, a composition of the proteome that - besides the produced target - is the same as the one of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coli . Importance The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial and error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli we show that optimizing its production is accompanied by alleviating stress. This indicates that monitoring stress responses could be used to facilitate enhancing recombinant protein production yields.