title: Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence.
abstract: The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.
authors.affiliation: Microbial Sciences Group, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
authors.forename: A
authors.initials: A
authors.lastname: Rozkov
authors.forename: C A
authors.initials: CA
authors.lastname: Avignone-Rossa
authors.forename: P F
authors.initials: PF
authors.lastname: Ertl
authors.forename: P
authors.initials: P
authors.lastname: Jones
authors.forename: R D
authors.initials: RD
authors.lastname: O'Kennedy
authors.forename: J J
authors.initials: JJ
authors.lastname: Smith
authors.forename: J W
authors.initials: JW
authors.lastname: Dale
authors.forename: M E
authors.initials: ME
authors.lastname: Bushell
mesh_terms: D001483:Base Sequence; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D006511:Hepatitis B Antigens / Q000235:genetics*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D006511:Hepatitis B Antigens