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Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

Lookup NU author(s): Dr Ronan O'Kennedy

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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.

Publication metadata

Author(s): Rozkov A, Avignone-Rossa CA, Ertl PF, Goryanin II, Jones P, O'Kennedy RD, Smith JJ, Dale JW, Bushell ME

Publication type: Article

Publication status: Published

Journal: Biotechnology and Bioengineering

Year: 2004

Volume: 88

Issue: 7

Pages: 909-915

ISSN (print): 0006-3592

ISSN (electronic): 1097-0290

Publisher: John Wiley & Sons, Inc.

URL: http://dx.doi.org/10.1002/bit.20327

DOI: 10.1002/bit.20327

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