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Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.Lactococcus lactis is a lactic acid bacterium that is used as a starter culture in the dairy industry. L. lactis has a rather simple and well-characterized metabolism and converts sugars mainly into lactic acid. In recent years L. lactis has also been evaluated as organism for production of industrial lactic acid (23, 38) and has been subjected to metabolic engineering to reroute its metabolism towards novel products (13,18,22,25,26). To take full advantage of bacterial processes, it is very important to understand which intracellular and extracellular factors influence the metabolic rate and product formation. L. lactis is an attractive model organism for studies of glycolysis and pyruvate metabolism, since oxidative phosphorylation normally does not occur and more than 90% of the carbon source is recovered as fermentation by-products, mainly lactate. In glycolysis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) converts NAD ϩ to NADH, which must be regenerated for continued carbon catabolism. Lactate dehydrogenase (LDH) regenerates NAD ϩ by converting the end product of glycolysis, pyruvate, to lactate. An alternative way for lactococci to regenerate NAD ϩ is by production of ethanol by alcohol dehydrogenase (ADH...

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Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.Lactococci are known for their homolactic metabolism, whereby more than 90% of the sugars present are converted into lactic acid. However, under certain conditions, the metabolism may shift to the production of mixed acids (acetate, ethanol, and formate). From the 1960s onward, the view that the control of this shift was modulated mainly by the intracellular concentration of fructose-1,6-biphosphate (FBP) activating both L-lactate dehydrogenase (L-LDH; EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40) was held (3,9,18,19). Inorganic phosphate (P i ) was recognized as a severe inhibitor of both enzymes. Apparently, both FBP and P i were seen to compete for the same allosteric site of LDH (9).Recently, this metabolic model was questioned by Garrigues et al. (5), who showed that the sugar metabolism of strain NCDO 2118 was instead regulated by the NADH/NAD ratio. A high ratio inhibited glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) and increased LDH activity. Many, but not all, researchers took up this view without reflection, creating a confusing situation. Currently, metabolic flux models based on enzyme kinetics are applied as predictive tools in metabolic engineering (see, e.g., reference 8), illustrating the importance of expressing kinetic characteristics adequately. Therefore, we undertook an investigation regarding the nature of the regulation of LDH activity among several Lactococcus lactis strains, including those frequently used in metabolic flux studies. We also examined the effects of ATP, ADP, AMP, and phosphoenolpyruvate (PEP), since Jonas et al. (9) observed strong competitive inhibition by ATP of LDH activity in L. lactis strain US3 (ϭNCIMB701197).L. lactis strains (listed in Table 1) were each grown anaerobically at 30°C in pH-controlled batch cultures on glucose (10 g/liter) with SD3 medium (17). The external pH was kept at 6.5, which corresponds to an internal pH of 7.2 (12). The cultures were harvested in the late exponential phase, centrifuged at 4°C for 10 min at 5,000 ϫ g, washed, and resuspended in triethanolamine buffer (50 mM triethanolamine [pH 7.2], 5 mM MgCl 2 ⅐ 6H 2 O). Cell extracts (CE) were prepared by using glass beads (17). LDH activity was measured spectrophotometrically by monitoring the oxidation of NADH (340 nm; ε ϭ 6,220 M Ϫ1 cm Ϫ1 ) at 30°C. One milliliter of the standard assay mixture consisted of triethanolamine buffer (50 mM; pH 7.2), NADH (0.3 mM), FBP (10 mM), and CE (approximately 160 mg of protein/liter). The reaction was initiated by the addition of pyruvate (initial concentration, 10 mM). One unit of LDH was de...