Heidi Winterberg Andersen

Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux

Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.

Quantifying the Importance of Regulatory Loops in homeostatic Control Mechanisms: Hierarchical Control of DNA Supercoiling

Many regulatory structures in the cell appear to aim at maintaining an optimal cellular state. These are here referred to as homeostatic control mechanisms. After a change in environmental conditions a cell adjusts to return as closely as possible to its previous state. Interestingly, these control mechanisms also function after perturbations of the cellular state itself. Apparently this type of regulatory mechanism is not so much addressed from the extracellular as it is from the intracellular environment, a topic dealt with in this volume in Chapter 26. Although the homeostatic control mechanisms are by definition beneficial to the biological system, they can be a nuisance when trying to engineer it. Attempts to manipulate the system tend to be largely overruled by subsequent adaptation. Part of such homeostatic control mechanisms is embedded in the nature of the kinetics of the catalytic steps in the system. On activation of such a step the product of the reaction will increase initially but this increase will lead to both an inhibition of the stimulated step and an activation of the subsequent step in the pathway. In this way, both enzymes surrounding the intermediate will tend to keep the concentration of the latter constant.