title: Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli.
abstract: BACKGROUND
  Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called 'metabolic burden': the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied.
  
  
  RESULTS
  Upon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration.
  
  
  CONCLUSIONS
  This work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production.
authors.affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. natalie.rahmen@avt.rwth-aachen.de.
authors.forename: Natalie
authors.initials: N
authors.lastname: Rahmen
authors.affiliation: Institute for Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52426, Jülich, Germany. a.fulton@fz-juelich.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fulton
authors.affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. nina.ihling@avt.rwth-aachen.de.
authors.forename: Nina
authors.initials: N
authors.lastname: Ihling
authors.affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. marzio.magni@avt.rwth-aachen.de.
authors.forename: Marzio
authors.initials: M
authors.lastname: Magni
authors.affiliation: Institute for Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52426, Jülich, Germany. karl-erich.jaeger@fz-juelich.de.|Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52426, Jülich, Germany. karl-erich.jaeger@fz-juelich.de.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. jochen.buechs@avt.rwth-aachen.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D018533:Biomass; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D002787:Sterol Esterase / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002787:Sterol Esterase; D010100:Oxygen