Is the Glycolytic Flux in Lactococcus lactisPrimarily Controlled by the Redox Charge?

Abstract

The involvement of nicotinamide adenine nucleotides (NAD ؉ , NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using 13 C and 31 P NMR to monitor in vivo the kinetics of the pools of NAD ؉ , NADH, ATP, inorganic phosphate (P i ), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of 13 C-labeled NAD ؉ and NADH. The capacity of L. lactis MG1363 to regenerate NAD ؉ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH ؊ deficient strain was constructed by double crossover. Upon supply of glucose, NAD ؉ was constant and maximal (ϳ5 mM) in the parent strain (MG1363) but decreased abruptly in the LDH ؊ strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH ؊ strain and NADH reached high levels (2.5 mM), reflecting severe limitation in regenerating NAD ؉ . However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mM. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P i content could play an important role.Lactococcus lactis plays an essential role in the manufacture of a wide range of dairy products. The relative simplicity of L. lactis metabolism that converts sugars via the glycolytic pathway to pyruvate, generating energy mainly through substrate level phosphorylation, makes it an attractive model organism to test metabolic engineering strategies. Moreover, the large number of genetic tools available for L. lactis (1) and the recent release of the complete genome sequence are additional incentives to study the physiology of this organism in great depth (2).Despite numerous studies, a satisfactory answer to the question, What controls the glycolytic flux in L. lactis? has not been put forward. During homolactic fermentation, regulation of the carbon flux has been associated with high levels of fructose 1,6-bisphosphate (FBP), 1 which activates lactate dehydrogenase (LDH; EC 1.1.1.27) and pyruvate kinase (PK; EC 2.7.1.40), directing the flux toward the production of lactate (3). A metabolic shift from homolactic (lactate production) to mixed acid fermentation (ethanol, acetate, and formate production) was observed in glucose-limited chemostat cultures (4). A deviation from homolactic fermentation was also reported under aerobic conditions (5) or during the metabolism of galactose (6). The format...

Catabolism of mannitol in Lactococcus lactis MG1363 and a mutant defective in lactate dehydrogenase

“…[1-"$C]Glucose (20 mM) or [1-"$C]mannitol (19 mM) were supplied to the cell suspension and the timecourse for their consumption, product formation and intracellular metabolite pools was monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, an NMR sample extract was prepared as reported previously (Neves et al, 1999(Neves et al, , 2002. The end products, lactate, acetoin, acetate, 2,3-butanediol, ethanol and formate, were quantified in the NMR sample extract by "H-NMR in a Bruker AMX300.…”

“…The end products, lactate, acetoin, acetate, 2,3-butanediol, ethanol and formate, were quantified in the NMR sample extract by "H-NMR in a Bruker AMX300. The concentration of intermediates that remained inside the cells (pyruvate, aspartate, succinate, alanine) was determined in fully relaxed "$C spectra of the NMR sample extracts as described by Neves et al (2002).…”

Natural sweetening of food products by engineering Lactococcus lactis for glucose production

“…The time course of lactose consumption, product formation, and changes in the pools of intracellular metabolites were monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, an NMR-sample extract was prepared as described previously (Neves et al, 1999;Neves et al, 2002). Carbon-13 spectra were acquired at 125.77 MHz on a Bruker DRX500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).…”

“…Lactate was quantified in the NMR-sample extract by 1 H-NMR in a Bruker AMX300 (Bruker BioSpin GmbH). The concentration of other metabolites (e.g., glucose) was determined in fully relaxed 13 C spectra of the NMR-sample extracts as described (Neves et al, 2002).…”

Subcellular metabolic organization in the context of dynamic energy budget and biochemical systems theories

“…In resting suspensions, meaning no-growth, metabolites were measured using NMR (Neves et al 2002b), while during growth, end-products and glucose were determined in supernatant samples by high-performance liquid chromatography.…”