A novel knock out strategy to enhance recombinant protein expression in Escherichia coli

Ashish K Sharma,Esha Shukla,Joseph Shiloach,Krishna J Mukherjee,Deepak S Janoti

doi:10.1186/s12934-020-01407-z

Abstract

BackgroundThe 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.ResultsWe 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 (Yp/x) was observed in a BW25113ΔcysJ knock out (Yp/x 0.57) and BW25113ΔelaA (Yp/x 0.49), whereas the Yp/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 (Yp/x 0.7) and BW25113ΔcysJΔyhbC (Yp/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.ConclusionCreating 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.

Highlights

The expression of recombinant proteins in E. coli triggers a cellular stress response (CSR) which mimics many features of the generalized stress response, the heat shock, the oxidative stress and the stringent response [1,2,3,4,5]
The CSR which is activated in response to the induction of recombinant protein expression can be seen as a feedback inhibition mechanism which operates at the global level to downregulate both growth and protein synthesis
A problem with this strategy is that it allows us to improve the expression of only a few genes while the CSR downregulates a large number of genes involved in the metabolic fluxes of several pathways

Summary

Introduction

The expression of recombinant proteins in E. coli triggers a cellular stress response (CSR) which mimics many features of the generalized stress response, the heat shock, the oxidative stress and the stringent response [1,2,3,4,5] This stress response (CSR) downregulates key metabolic pathways of the central carbon metabolism, ATP synthesis, substrate uptake, ribosomal synthesis as well as activating the glucose overflow mechanism leading to acetate formation [1, 3, 6]. Increasing expression of downregulated genes such as prsA and glpF helped in enhancing the expression levels of IGF-If from 1.8 to 4.3 g/l [23] These strategies underscore the importance of shifting our focus away from the individual steps of protein synthesis towards a more global approach of identifying key factors in recombinant protein production. 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

Methods

Results

Conclusion