Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae

Abstract

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

“…These studies on PFK-1 overexpression in CHO-K1 cells are in accordance, therefore, with the previous studies in S. cere isiae, A. niger and S. tuberosum [7][8][9], which showed that overexpression of PFK-1 had little or no effect on glycolytic flux, and with mathematical modelling studies of erythrocyte glycolysis which indicated that PFK-1 has a relatively low flux control coefficient [14]. These data are also in agreement with the more general finding that overexpression of an individual enzyme in any metabolic pathway is unlikely to have a significant effect on pathway flux [10,44].…”

“…This finding accords with similar studies on the overexpression of PFK-1 in other systems [7][8][9], and the finding that, in order to obtain a significant increase in flux in a metabolic pathway, the activity of several enzymes must be increased [10,45]. This is also consistent with the expectation that flux will not increase unless there is an increased demand for glycolytically derived ATP or for glycolytic intermediates.…”

“…Furthermore, selective increases in the expression of the enzyme in a variety of organisms, using molecular genetic techniques, has failed to produce significant increases in glycolytic flux. These include studies in the yeast Saccharomyces cere isiae [7], in the filamentous fungus Aspergillus niger [8] and in the potato (Solanum tuberosum) tuber [9]. These latter studies are in accordance with many other studies which have shown that increasing the activity of a single enzyme in a metabolic pathway will only very rarely have a significant effect on flux [10].…”

“…In yeast there was, instead, a decrease in the steady-state level of the allosteric activator of PFK-1, fructose 2,6-bisphosphate [7]. We suggested that this would decrease the specific activity of the enzyme, thus compensating, at least semi-quantitatively, for the increased concentration of the enzyme, and that this accounted for the absence of a change in flux through the enzyme and in the fructose 1,6-bisphosphate concentration.…”

“…In yeast we did observe a small but significant increase in glycolytic flux when we overexpressed PFK-1 in aerobic cells. However, this occurred at the expense of a decrease in oxygen consumption, such that the overall calculated rate of ATP turnover in the cell remained unchanged [7].…”