Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor.

A first study of the in vivo kinetic properties of primary metabolism of Penicillium chrysogenum is presented. Dynamic metabolite data have been generated by rapidly increasing the extracellular glucose concentration of cells cultivated under well-defined conditions in an aerobic glucose-limited chemostat followed by measurement of the fast dynamic response of the primary metabolite levels (glucose pulse experiment). These experiments were carried out directly in the chemostat as well as in a mini plug flow reactor (BioScope) outside the chemostat. The results of the glucose pulse experiments carried out in the chemostat and the Bioscope were highly similar. During the 90 s time window of the pulse experiment, the glucose consumption rate increased to a value twice as high as in the steady state, a much lower increase than observed for the fermenting yeast Saccharomyces cerevisiae under similar conditions. Although the observed metabolite patterns in P. chrysogenum were comparable to S. cerevisiae large differences in the magnitude of the dynamic behavior were observed between both organisms. During the pulse experiment the level of glycolytic and TCA cycle intermediates, and adenine nucleotides changed between two- and five-fold. Furthermore, a highly similar five-fold increase in the cytocolic NADH/NAD ratio could be calculated from two independent equilibrium assumptions (fructose 1,6 bis-phosphate to the pool of 2 and 3PG and oxaloacetate to fumarate with glutamate transaminase). It was also found that the C4 pool (aspartate, fumarate, and malate) became much more reduced due to this increase in NADH/NAD ratio. Equilibrium conditions were confirmed to exist in the hexose-P pool, the glycolysis between F16bP and 2+3PG and in the C4 pool of the TCA cycle (fumarate, malate, oxaloacetate and aspartate).