title: Metabolomic analysis of riboswitch containing E. coli recombinant expression system.
abstract: In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional-translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Howbeer
authors.initials: H
authors.lastname: Muhamadali
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. neil.dixon@manchester.ac.uk.
authors.forename: Rosa
authors.initials: R
authors.lastname: Morra
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Drupad K
authors.initials: DK
authors.lastname: Trivedi
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Nicholas J W
authors.initials: NJ
authors.lastname: Rattray
authors.affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. neil.dixon@manchester.ac.uk.
authors.forename: Neil
authors.initials: N
authors.lastname: Dixon
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D004734:Energy Metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D055432:Metabolomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D058928:Riboswitch; D015533:Transcriptional Activation
chemical_list: D011994:Recombinant Proteins; D058928:Riboswitch; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins