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 o peron, harboring pfk, pyk, and ldh, were replaced by synthetic promoters we re constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kin ase and lactate dehydrogenase remained closer to the wild-type level. In de fined 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 reduce d to 62 to 76% of wild-type levels. In complex medium growth was even furth er reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-li mited conditions, One explanation could be that the reduced activity of pho sphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemosta t, the growth rate could gradually be increased to 195% of the growth fate observed in glucose-saturated batch culture, suggesting that phosphofructok inase does affect the concentration of upstream metabolites. The pools of g lucose-6-phosphate and fructose-6-phosphate were subsequently found to be i ncreased two- to fourfold in the las mutants, which indicates that phosphof ructokinase exerts strong control over the concentration of these metabolit es.