Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures
Pasini, Martina (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Fernández-Castané, Alfred (Aston University. Aston Institute of Materials Research)
Caminal i Saperas, Glòria (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Overton, Tim W. (University of Birmingham. College of Engineering and Physical Sciences)
Ferrer, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Fecha:	2022
Resumen:	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.
Ayudas:	Ministerio de Ciencia e Innovación CTQ2011-28398-CO2-01 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-281
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Materia:	High-cell-density fed-batch cultures ; Recombinant protein production ; Antibiotic-free expression system ; Escherichia coli ; Bioprocess optimization
Publicado en:	Journal of Industrial Microbiology and Biotechnology, Vol. 49, Issue 4 (July 2022) , art. kuac018

DOI: 10.1093/jimb/kuac018
PMID: 35657374

11 p, 1.6 MB

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