Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli

Abstract

Background A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. Results We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. Conclusion The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

“…The results showed that the double knockout mutant BW25113ΔelaA + ΔcysW (DKO) had the highest activity in asparaginase production with 70.3 units/ml. To further unravel the mechanisms involved in CSR mitigation by the DKO strain, Guleria et al [65] used the strain to overexpress the Rubella E1 gene and performed a transcriptome analysis. Compared to the wild type, down-regulation of multiple genes related to growth-critical processes was suppressed in the DKO strain, including translation, transcription, RNA and ribosome biogenesis, transport, energy metabolism and other catabolic processes.…”

CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli

“…In Escherichia coli , it has been shown that co-expression of critical factors such as chaperones can unburden the metabolic load which helps to increase the product yield when folding and solubility is crucial ( 6–9 ). In contrast, down-regulation or even knockouts of proteases provides better stability of the product ( 4 , 10–12 ). However, the specific factors that improve the production process performance are manifold and largely unknown.…”