Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture.

BACKGROUND: Despite the fact that many reports deal with glycolysis in Lactococcus lactis, there is not much information on the regulation of uptake of glucose itself. The aim of the present work was to investigate the effect of the glucose level on its specific uptake rate. RESULTS: Studies on aeration levels in pH controlled L. lactis spp. lactis batch cultures on glucose (55 mM) showed that product formation is extremely homolactic and the highest yield of lactate on glucose is obtained under microaerobic conditions (5% dissolved oxygen). Microaerobic conditions were therefore applied in experiments carried out to investigate the regulation of the uptake of glucose. The tool of glucostat fed-batch culture was employed, in which glucose was added at a rate suitable to maintain a stable concentration throughout the runs with glucose concentration ranging from 13.75 to 555 mM. The glucostat experiments showed that the concentration of glucose influences its specific uptake rate and consequently the glycolytic flux, as well as the fermentation pattern. The highest specific activities of the key glycolytic enzymes PFK, PYK and the LDH were obtained at 55 mM glucose, the area of the highest observed glycolytic flux. Reduction of the glycolytic flux by 55% in the 277 mM glucostat corresponded to an almost identical reduction in PFK activity, indicating a certain controlling influence of this enzyme on the flux, through the glucose effect. CONCLUSION: Determination of intracellular metabolites' pools showed that FBP cannot be regarded as a direct regulator of product formation, since almost identical concentrations were obtained at both low (13.75 mM) and high (138 mM) glucose levels, at which neither the glucose uptake rates and the glycolytic flux, nor the fermentation patterns were similar (mixed acids vs homolactic, respectively). Glucostat data showed instead that the control of the flux through the glycolytic pathway under the examined conditions, resides to a large extent in processes outside the pathway, like the ATP consuming reactions and glucose transport. A regulation mechanism is proposed governed by the energy state of the cell by which L. lactis can handle the glycolytic flux through the allosteric properties of key enzymes, with PFK having a significant influence on the control.