Metabolic approaches for the optimisation of recombinant fermentation processes

The aim of this work was the establishment of a novel method to determine t he metabolic load on host-cell metabolism resulting from recombinant protei n production in Escherichia coli. This tool can be used to develop strategi es to optimise recombinant fermentation processes through adjustment of rec ombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosp hate (ppGpp), and its precursor nucleotides were selected for the estimatio n of the metabolic load relating to recombinant-protein production. An impr oved analytical method for the quantification of nucleotides by ion-pair, h igh-performance liquid chromatography was established. The host-cell respon se upon overexpression of recombinant protein in fed-batch fermentations wa s investigated using the production of human superoxide dismutase (rhSOD) a s a model system. E. coli strains with different recombinant systems (the T 7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.