Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro 31 P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.Lactic acid bacteria (LAB) employ a few glycolytic pathways to funnel carbohydrates into the common three-carbon intermediate stage of glycolysis. In general, homofermentative LAB convert carbohydrates into lactate using the Embden-Meyerhof pathway (EMP), whereas heterofermentative LAB produce lactate, ethanol, and carbon dioxide using the phosphoketolase pathway (PKP) (8). The PKP is usually used by LAB to ferment pentoses (9), and it has a poor energy yield compared to that of the EMP (16). This disadvantage can be compensated for by the addition of external electron acceptors, which creates alternative pathways for NAD(P)H reoxidation and may stimulate growth (33). A part of the acetyl phosphate can then be converted into acetate instead of ethanol, thereby gaining one additional ATP, making the PKP as efficient as the EMP.Lactobacillus reuteri ATCC 55730 has been described as a heterofermentative bacterium using the PKP (9) for converting glucose to lactate, ethanol, and CO 2 . Sucrose is converted into lactate, acetate, ethanol, CO 2 , and mannitol with this strain, whereby the latter is formed from the fructose half that functions solely as an electron acceptor (2).Heterofermentative lactobacilli take up sugars by nonphosphorylating permease systems, but several possess the phosphotransferase system for the uptake of fructose, which is inevitably connected to a fully operating EMP instead of the PKP because of the requirement of the production of two phosphoenolpyruvates (PEP) per sugar unit (18, 25).In the current study, we describe and s...

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