Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

Abstract

A series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C J F1 LDH ¼ 2 1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate dehydrogenase, only 70% more than needed to catalyze the lactate flux in the wild-type cells.Keywords: lactic acid bacteria; metabolic control analysis; gene expression; fermentation.Lactococcus lactis plays an important role in dairy fermentations, mainly in the production of cheeses. In these fermentation processes, lactose is present at high concentrations (50 g·L 21 ) and is converted through glycolysis to lactic acid, with minor amounts of other compounds being produced in addition (homolactic fermentation). The resulting low pH contributes to the texture and flavor of cheeses and inhibits the growth of other bacterial species. Under conditions where sugar becomes limiting for growth of Lactococcus lactis, the metabolism shifts to mixed-acid products, i.e. formate, acetate and ethanol along with smaller amounts of lactate [1,2].Work has been performed in the past to study the mechanisms involved in the shift between the two different fermentation modes in L. lactis. In the presence of excess sugar, the concentration of fructose 1,6-bisphosphate, the triose-phosphates, pyruvate, and the NADH/NAD þ ratio are high, whereas the concentration of phosphoenolpyruvate and inorganic phosphate are relative low [3 -6]. In contrast, when sugar is limiting the concentration of these metabolites and cofactors are reversed to the opposite, high or low level. Particularly, the level of fructose-1,6-bisphosphate, which is known to activate both pyruvate kinase and lactate dehydrogenase, has been suggested to play a key role in the regulation of the fermentation mode [1].Work has also been performed to determine the factors that control the flux through glycolysis by applying metabolic control analysis [7,8]. Based on inhibitor titration, Poolman et al. [9] suggested that glyceraldehyde 3-phosphate dehydrogenase had a large amount of control ove...

IS 981 -Mediated Adaptive Evolution Recovers Lactate Production by ldhB Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of Lactococcus lactis

“…The inferior characteristics observed for the Ldh enzyme present in this strain are reflected in the observation that the lactateproducing capacity in this strain never exceeded approximately 85% of the total carbon flux, which is significantly lower than that observed in wild-type cells (Ͼ95%) (20,43). Previously, it has been shown that the las operon-encoded Ldh enzyme has a low degree of control over lactate formation rates in L. lactis (1), which is in agreement with the metabolic predictions generated by the kinetic model of the lactococcal pyruvate metabolism (23). However, replacement of the las operon-encoded Ldh enzyme with LdhB in that same model confirms the metabolic values reported here (data not shown; reference 23; jjj .biochem.sun.ac.za/wcfs.html).…”

Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

“…Quantification of glucose, pyruvate, lactate, formate, acetoin, acetate, and ethanol by HPLC was performed as described previously (1).…”

“…Andersen and colleagues used modulation of gene expression to show that lactate dehydrogenase has no control on the glycolytic flux in L. lactis MG1363 (1). Phosphofructokinase (PFK), on the other hand, appeared to be present in a very low excess, and even a small reduction of PFK activity resulted in an almost proportional decrease in the glycolytic flux (2).…”

The las Enzymes Control Pyruvate Metabolism in Lactococcus lactis during Growth on Maltose

“…The activities of PYK and LDH have previously been shown to affect pyruvate metabolism in L. lactis (1,7), and differences in fermentation patterns between glucose and maltose could thus be due to differences in the specific activities of these enzymes on maltose. The specific activities of the three las enzymes in wild-type strain MG1363 grown on maltose and on glucose were therefore measured and found to be almost identical (data not shown).…”

“…One study focusing on the las operon, coding for phosphofructokinase (PFK), pyruvate kinase (PYK), and LDH (9), revealed that LDH has a strong negative effect on the mixed acid flux; the flux control coefficient was less than Ϫ1, meaning that a 10% reduction in LDH leads to a Ͼ10% increase in the mixed acid flux (1). Another study revealed that PYK exhibits strong positive control of formate and acetate production (flux control coefficient for PYK on the formate and acetate flux were both close to 1) (7).…”