On-line estimation of the metabolic burden resulting from the synthesis ofplasmid-encoded and heat-shock proteins by monitoring respiratory energy generation

Human basic fibroblast growth factor (hFGF-2) was produced in high-cell den sity cultures of recombinant Escherichia coli using a temperature-inducible expression system. The synthesis rates of proteins were followed by two-di mensional gel electrophoresis of the S-35-methionine-labeled proteom. After temperature induction of hFGF-2 synthesis, the rate of total protein synth esis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins. The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 ge ne but followed time profiles different from the producing strain. The ener gy demand for the extra synthesis of plasmid-encoded and heat-shock protein s resulted in an elevated respiratory activity and, consequently, in a redu ction of the growth rate and the biomass yield. A procedure was developed t o relate the energy demand for the additional synthesis of these proteins t o the generation of energy in the respiratory pathway. Specific energy prod uction was estimated based on on-line measurable rates of oxygen consumptio n, or carbondioxide evolution and growth, respectively. In this way, the me tabolic burden resulting from the synthesis of plasmid-encoded and heat-sho ck proteins was quantified from on-line accessible data. (C) 2001 John Wile y & Sons, Inc.