KINETIC, DYNAMIC, AND PATHWAY STUDIES OF GLYCEROL METABOLISM BY KLEBSIELLA-PNEUMONIAE IN ANAEROBIC CONTINUOUS-CULTURE - III - ENZYMES AND FLUXES OF GLYCEROL DISSIMILATION AND 1,3-PROPANEDIOL FORMATION

The initial steps of glycerol dissimilation and 1,3-propanediol (1,3-P D) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enz ymes in continuous culture under conditions of steady state and oscill ation and during transient phases. The enzymes studied included glycer ol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3-propanedi ol oxidoreductase (PDOR). Three conclusions can be drawn from the stea dy-state results. First, glycerol concentration in the culture is a ke y parameter that inversely affects the in vitro activities (concentrat ions) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro a nd in vivo enzyme activities under low glycerol concentrations, but no t under glycerol excess. Second, whereas the flux through the oxidativ e pathway of glycerol dissimilation is governed mainly by the regulati on of in vivo enzyme activity on a metabolic level, the flux through t he reductive pathway is largely controlled by the synthesis of enzymes . Third, GDHt is a major rate-liming enzyme for the consumption of gly cerol and the formation of 1,3-PD in K. pneumoniae at high glycerol co ncentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average va lues of the in vitro activities during an oscillation cycle agreed wel l with their corresponding values for nonoscillating cultures under si milar environmental conditions. Experiments with step changes in the f eed concentration of glycerol demonstrated that growth and product for mation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the gene tic and metabolic networks. They should be considered when modeling th e dynamics of the culture and attempting to improve the formation of 1 ,3-PD. (C) 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 544-552, 1998.