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

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 impro...

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Autores principales: Sharma, Ashish K., Shukla, Esha, Janoti, Deepak S., Mukherjee, Krishna J., Shiloach, Joseph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376861/
https://www.ncbi.nlm.nih.gov/pubmed/32703203
http://dx.doi.org/10.1186/s12934-020-01407-z
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author Sharma, Ashish K.
Shukla, Esha
Janoti, Deepak S.
Mukherjee, Krishna J.
Shiloach, Joseph
author_facet Sharma, Ashish K.
Shukla, Esha
Janoti, Deepak S.
Mukherjee, Krishna J.
Shiloach, Joseph
author_sort Sharma, Ashish K.
collection PubMed
description 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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spelling pubmed-73768612020-07-23 A novel knock out strategy to enhance recombinant protein expression in Escherichia coli Sharma, Ashish K. Shukla, Esha Janoti, Deepak S. Mukherjee, Krishna J. Shiloach, Joseph Microb Cell Fact Research 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. BioMed Central 2020-07-23 /pmc/articles/PMC7376861/ /pubmed/32703203 http://dx.doi.org/10.1186/s12934-020-01407-z Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Sharma, Ashish K.
Shukla, Esha
Janoti, Deepak S.
Mukherjee, Krishna J.
Shiloach, Joseph
A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title_full A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title_fullStr A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title_full_unstemmed A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title_short A novel knock out strategy to enhance recombinant protein expression in Escherichia coli
title_sort novel knock out strategy to enhance recombinant protein expression in escherichia coli
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376861/
https://www.ncbi.nlm.nih.gov/pubmed/32703203
http://dx.doi.org/10.1186/s12934-020-01407-z
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