Background: The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression. Results: We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Y p/x) was observed in a BW25113ΔcysJ knock out (Y p/x 0.57) and BW25113ΔelaA (Y p/x 0.49), whereas the Y p/x of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Y p/x 0.7) and BW25113ΔcysJΔyhbC (Y p/x 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium. Conclusion: Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.

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Background: The introduction of engineered cysteine in staphylococcal protein A (SPA-cys) for site-specific conjugation results in a substantial amount of dimerized SPA due to spontaneous oxidation during its production, leading to inaccessibility and thus rendering it unusable. Monomers are usually recovered from dimers by using reducing agents before conjugation in subsequent steps. However, this leads to low conjugation efficiency and increases overall cost and production time. This study aims to systematically compare and quantify the monomeric and dimeric content of SPA when produced through intracellular and extracellular routes in E. coli. Methods: Purified SPAs with and without cysteine from both intracellular and extracellular processes are compared for their monomeric content and efficiency to conjugate on solid support matrix with and without an additional pre-step of reduction. Results: The monomeric form of SPA-cys, which is a desired key quality attribute, is less than 50% when produced extracellularly. SPA-cys produced through the intracellular production process has high monomeric content (≥85%) and shows higher binding to solid support. Conclusion: The study demonstrates that the intracellular route for production of SPA-cys should be the preferred method, and the release assays for SPA-cys products should include the amount of monomeric content as one of the quality attributes. The abundance of monomeric content enhances the site-specific conjugation efficiency and density of SPA on the resin matrix.