Presently an increasing gap is developing between our experimental capabilities in metabolic engineering of microbial metabolism and our quantitative theoretical understanding of the kinetic interaction between primary metabolism and product pathways. Such theoretical understanding is absolutely needed for a rational design of said metabolic engineering experiments and targets. To obtain such understanding in-vivo kinetic experiments and in-vivo kinetic models are needed. To this end the following new methods for in-vivo kinetic studies of Saccharomyces cerevisiae have recently been developed. The Bioscope device allows the reliable and repeated perturbation (e.g. glucose pulse, or inhibitor etc.) of steady state biomass outside the fermentor and subsequent sampling and quenching to measure glycolytical intermediates and nucleotides in a time frame of 0-70 seconds. Dynamic modelling of fermentor off-gas O2/CO2-measurements allows to calculate O2 uptake and CO2 production rates in such a perturbation experiment (0-70 seconds time windows). A new LC-MSMS based method has been developed to measure large sets of intracellular metabolites in said in-vivo kinetic experiments. It has been shown for the first time that in long chemostat cultivation (up to 800 hrs.) intracellular metabolites levels drop, showing absence of a real steady state. A new kinetic format, lin log kinetics, has been developed for describing the intracellular kinetic behavior of metabolic networks. This format allows general analytical solutions of networks flux, metabolic levels. From simulation studies it appears that this approach is remarkable accurate in describing intracellular metabolite dynamics and in metabolic design questions of where to change enzyme levels and how much to achieve a desired change in fluxes and metabolite levels. At this moment these tools are being applied in metabolic engineering studies of Saccharomyces cerevisiae and Penicillin chrysogenum.
|Journal||Mededelingen - Faculteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen - Universiteit Gent|
|Issue number||3 a|
|Publication status||Published - 2001|