Metabolic approaches for the optimisation of recombinant fermentation processes.
The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 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.
Cserjan-Puschmann
M
M
Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Austria.
Kramer
W
W
Duerrschmid
E
E
Striedner
G
G
Bayer
K
K
D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D009711:Nucleotides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013482:Superoxide Dismutase / Q000096:biosynthesis
D009711:Nucleotides; D011994:Recombinant Proteins; D013482:Superoxide Dismutase