Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures
| Autor: | Martina Pasini, Alfred Fernández-Castané, Gloria Caminal, Tim W Overton, Pau Ferrer |
|---|---|
| Přispěvatelé: | Ministerio de Ciencia e Innovación (España) |
| Rok vydání: | 2022 |
| Předmět: |
Bioengineering
Antibiotic-free expression system Applied Microbiology and Biotechnology Recombinant protein production Recombinant Proteins Anti-Bacterial Agents Phosphates Bioprocess optimization High-cell-density fed-batch cultures Batch Cell Culture Techniques Fructose-Bisphosphate Aldolase Escherichia coli Biotechnology Aldehyde-Lyases Plasmids |
| Zdroj: | Dipòsit Digital de Documents de la UAB Universitat Autònoma de Barcelona Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1476-5535 |
| Popis: | 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. This work was supported by the Spanish MICINN, project number CTQ2011-28398-CO2-01, the research group 2009SGR281, and by the Bioprocess Engineering and Applied Biocatalisys Group, Department of Chemical, Biological and Environmental Engineering of the Universitat Autònoma de Barcelona, Cerdanyola del Valles (Spain). |
| Databáze: | OpenAIRE |
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