Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.

FIG. 1.

Relative activities of GAPDH for clones with altered expression of gapB. GAPDH activity was measured in extracts from strains with (i) an extra copy of gapB inserted in the L. lactis chromosome in the attachment site for phage TP901-1 and transcribed from various synthetic promoters (black bars) or (ii) the native gapB promoter replaced by a library of synthetic promoters in the L. lactis MG1363 chromosome (gray bars). The specific GAPDH activity in MG1363 was determined to 1.48 U/mg of protein.

FIG. 2.

Effect of GAPDH activity on growth (A) and glycolytic flux (B) of L. lactis, relative turnover number of GAPDH (C), and flux control by GAPDH (D). The relative turnover number of GAPDH was calculated as the ratio of the relative glycolytic flux and the relative GAPDH enzyme activity and plotted as a function of the relative GAPDH activity. Curve fitting is described in Materials and Methods.

FIG. 3.

Effect of changes in GAPDH activity on the glycolytic flux in resting cells. See Materials and Methods and Results for details. The curve fitted to the experimental data points for the flux in growing cells is shown for comparison.

FIG. 4.

Glycolytic flux in strains with increased GAPDH activity resulting from overexpression of gapA. The glycolytic fluxes are plotted as functions of the respective GAPDH activities of strains MG1363, CS267, and CS314.

FIG. 5.

Relative lactate and formate production of strains with altered GAPDH activity from an anaerobic batch experiment at 30°C in SA medium supplemented with 1.2 g of glucose per liter.