Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence.
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.
Rozkov
A
A
Microbial Sciences Group, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
Avignone-Rossa
C A
CA
Ertl
P F
PF
Jones
P
P
O'Kennedy
R D
RD
Smith
J J
JJ
Dale
J W
JW
Bushell
M E
ME
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*
D029968:Escherichia coli Proteins; D006511:Hepatitis B Antigens