Metabolic flux analysis of recombinant protein overproduction in Escherichia coli

A metabolic model for cell growth and recombinant protein overproduction in Escherichia coli, which includes expression vector properties such as plasmid copy number and promoter strength as input parameters, was developed. Linear programming was used to obtain pathway fluxes for selected systems. The experimentally observed slow growth of recombinant E. coliXL1 in minimal medium supplemented with glucose accompanied with significant acetic acid secretion was successfully simulated by metabolic flux analysis (MFA) computations. The expression of the fusion protein GI-malE induced by IPTG caused a severe retardation in cell growth rate from mu = 0.066 to 0.006 h(-1) and increased acetic acid secretion to the medium. The metabolic flux analysis of this transition in growth condition show that cells shift the usage of the available substrate from anabolic to catabolic pathways to increase their ability to produce energy for plasmid-encoded protein synthesis, stress protein synthesis and maintenance requirements. The increase in catabolic fluxes led to increased acetic acid secretion rate. MFA analysis of EcoRI endonuclease expression in E. coli 294 at high growth rate and accompanied by low growth rate depression showed that the flux distribution was very similar for both host and induced cells. The pathway utilization simulate exponential growth condition with preference for the PP pathway and activation of the glyoxalate shunt to meet the high demand for NADPH, NADH and biosynthetic intermediates at high growth rate. For both of the systems studied the pathway utilization and relative flux distributions obtained from MFA are in good agreement with reported E. coli gene expression profiles at different stages of growth and during recombinant protein overexpression. (C) 2004 Elsevier B.V. All rights reserved.