¹³ C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

Abstract

13 C nuclear magnetic resonance ( 13 C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4- 13 C]citrate, [3- 13 C]pyruvate, and [2- 13 C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. α-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the α-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.

Metabolic flux in glucose/citrate co-fermentation by lactic acid bacteria as measured by isotopic ratio analysis

“…By this indirect means, it can be estimated that, in their fermentation conditions, the incorporations from glucose into both lactic acid and acetoin were about half those found in the present study. As, however, the concentration ratio in [6] is 2:1 in favor of citrate while in the present study it is 2:1 in favor of glucose, it is perhaps not surprising that glucose was more strongly incorporated in the present study than in [6]. It is clear that the degree of incorporation of the two carbon sources into the aroma compounds might vary markedly with the fermentation conditions used.…”

“…Thus, there is no evidence that during fermentation in standard M17 broth with an initial pH of 6.2^6.4 citrate is the preferred source of carbon for diacetyl and acetoin biosynthesis. The proportions of carbon incorporation have previously [6] been estimated from the molar ratios of 13 C incorporation (determined by 13 C/ 1 H heteronuclear coupling) in growing cells (initial pH 6.4) provided with citrate (17.5 mM) and glucose (8.8 mM). By this indirect means, it can be estimated that, in their fermentation conditions, the incorporations from glucose into both lactic acid and acetoin were about half those found in the present study.…”

“…lactis 19B, it was found that at pH 5.5, 50^70% of available citrate was converted to lactic acid [2]. Furthermore, in both non-growing and growing cultures (initial pH 6.4), while citrate alone was substantially converted to acetoin, the co-metabolism of citrate with glucose led to up to 80% bioconversion of citrate to lactic acid [6]. These studies have several major drawbacks.…”

Malolactic fermentation in wine - beyond deacidification

“…This co-metabolism of citrate-glucose has been shown to enhance the growth rate and biomass yield of this bacterium, which result from increased ATP synthesis both by substrate-level phosphorylation via acetate kinase and by a chemiosmotic mechanism (proton motive force) (Salou et al 1994;Ramos and Santos 1996). The growth stimulation of citrate-sugar co-fermentation by the same mechanisms has also been reported in citrate-fermenting dairy LAB (Cogan 1987;Schmitt and Diviès 1991;Ramos et al 1994;Bandell et al 1998). From a winemaker's point of view, the co-metabolism of citrate-sugar increases the formation of the volatile acid (acetate) in wine, which can affect the wine aroma detrimentally if present at excessive levels.…”

Natural‐abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose–citrate cofermentation by Lactococcus lactis

“…results in diacetyl accumulation has received considerable interest [14,15] and, as has been previously argued [11], is complex. What is apparent is that the cometabolism of citrate and glucose leads to the enhanced production of these compounds by re-routeing of the metabolic throughput [3,[7][8][9]16,17]. Because this is not specifically due to the metabolism of citrate to the C4 compounds [10,11], it must reflect an overall shift in the balance between different routes for pyruvate catabolism.…”