Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux

Abstract

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.

FIG. 1

Strategy used to alter expression of las genes. (A) Schematic presentation of the chromosomal las operon in MG1363 and of the DNA fragments which have been cloned in pHWA182. The bent arrow indicates the native promoter P_las of the operon. The heavy black line indicates the DNA fragment upstream of the operon. The truncated pfk region is indicated by the light gray line. (B) The synthetic promoter CP25 was flanked by the upstream region of the las operon and the truncated region of pfk on the E. coli vector pHWA182. (C) After selection for erythromycin resistance and subsequent counterselection with ampicillin, the native promoter P_las was replaced by CP25 by double homologous recombination between pHWA182 and MG1363, resulting in HWA217.

FIG. 2

Growth curves of las mutants HWA217 and HWA232 and wild-type control strain MG1363. The cultures were grown as batch fermentation cultures in SA medium supplemented with 0.25% glucose, and pH was kept constant at 6.8 by the addition of 2 M NaOH.

FIG. 3

End-product formation of las mutants. The products produced by the las mutants and MG1363 during batch fermentation were analyzed by HPLC. The strains displayed a homolactic fermentation pattern. Samples were withdrawn from cultures in the stationary growth phase with an OD₆₀₀ of approximately 1.1, immediately after the glucose had been exhausted. The C-mole recovery ranged between 80 and 83%.

FIG. 4

Growth of las mutant HWA217 and MG1363 in chemostat culture at various dilution rates. Biomass concentration of the cultures is shown during the experiments as a function of the dilution rate. Growth was initiated in a batch fermentation. Before the cultures entered the stationary growth phase, the chemostat was inoculated at a dilution rate of 0.40 h⁻¹. The experiments in which the dilution rate was increased rapidly (experiment A) or slowly (experiment B) are shown with closed or open symbols, respectively. The biomass concentrations of MG1363 and HWA217 are shown as squares and circles, respectively. The solid line indicates the biomass concentration of HWA217 when the dilution rate was increased slowly. This line also shows the biomass concentration of MG1363 in both cases. The broken line indicates the biomass concentration of HWA217 when the dilution rate was increased rapidly. The cultures were grown in M17 supplemented with 0.25% glucose, and pH was kept constant at 6.8 by the addition of 2 M NaOH.