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

Content Provider	Springer Nature : BioMed Central
Author	Guleria, Richa Jain, Priyanka Verma, Madhulika Mukherjee, Krishna J.
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.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-020-01488-w.pdf
Ending Page	17
Page Count	17
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/s12934-020-01488-w
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	19
Language	English
Publisher	BioMed Central
Publisher Date	2020-12-11
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Escherichia coli Cellular stress response Knockouts Recombinant protein expression Signaling Transcriptome
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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