Metabolic approaches for the optimisation of recombinant fermentation processes |
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Authors: | M Cserjan-Puschmann W Kramer E Duerrschmid G Striedner K Bayer |
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Institution: | (1) Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria e-mail: bayer@mail.boku.ac.uk Tel.: +43-1-36006-6220 Fax: +43-1-3697615, AT |
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Abstract: | 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.
Received: 30 April 1999 / Received revision: 26 July 1999 / Accepted: 1 August 1999 |
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