C-13 NUCLEAR-MAGNETIC-RESONANCE STUDIES OF CITRATE AND GLUCOSE COMETABOLISM BY LACTOCOCCUS-LACTIS

C-13 nuclear magnetic resonance (C-13-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongr owing cells of Lactococcus lactis subsp. lactis 19B under anaerobic co nditions. The metabolism of citrate plus glucose during growth was als o monitored directly by in vivo NMR. Although pyruvate is a common int ermediate metabolite in the metabolic pathways of both citrate and glu cose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-C-13] c itrate, [3-C-13]pyruvate, and [2-C-13]glucose. The presence of an addi tional substrate caused a considerable stimulation in the rates of sub strate utilization, and the pattern of end products was changed. Aceta te 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 gluco se on the metabolism of citrate by dilute cell suspensions was also pr obed by using analytical methods other than NMR. Pyruvate dehydrogenas e (but not pyruvate formate-lyase) was active in the conversion of pyr uvate to acetyl coenzyme A. alpha-Acetolactate was detected as an inte rmediate metabolite of citrate or pyruvate metabolism, and the labelin g pattern of the end products agrees with the alpha-acetolactate pathw ay. 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. lact is is presented.