Process Intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

Abstract

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Publication DOI: https://doi.org/10.1093/jimb/kuac018
Divisions: College of Engineering & Physical Sciences > School of Infrastructure and Sustainable Engineering > Chemical Engineering & Applied Chemistry
Additional Information: © The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.
Uncontrolled Keywords: Aldehyde-Lyases/genetics,Anti-Bacterial Agents/metabolism,Batch Cell Culture Techniques,Escherichia coli/genetics,Fructose-Bisphosphate Aldolase/genetics,Phosphates/metabolism,Plasmids/genetics,Recombinant Proteins/metabolism
Publication ISSN: 1476-5535
Full Text Link:
Related URLs: https://academi ... 392?login=false (Publisher URL)
PURE Output Type: Article
Published Date: 2022-07
Published Online Date: 2022-06-03
Accepted Date: 2022-05-05
Authors: Pasini, Martina
Fernandez-Castane, Alfred (ORCID Profile 0000-0002-2572-7797)
Caminal, Gloria
Overton, Tim W.
Ferrer, Pau

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