Effect of different NADH oxidase levels on glucose metabolism by Lactococcus lactis: kinetics of intracellular metabolite pools determined by in vivo nuclear magnetic resonance

Abstract

Three isogenic strains of Lactococcus lactis with different levels of H(2)O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX(-) strain harboring a deletion of the gene coding for H(2)O-forming NADH oxidase, and a NOX(+) strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using (13)C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P(2)] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P(2) depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX(-) strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX(+) strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX(-) and parent strains; in the NOX(+) strain the carbon flux was diverted to the production of alpha-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.

FIG. 1.

Kinetics of [6-¹³C]glucose consumption and product formation (A and B) and pools of intracellular metabolites (C and D) in MG1363 under an oxygen atmosphere (A and C) or under anaerobic conditions (B and D) as monitored by ¹³C-NMR. For anaerobic studies, cells were grown in deaerated chemically defined medium, while aerobic growth was achieved by maintaining a constant air pressure of 80% in the bioreactor vessel. Symbols: ⧫, glucose; □, lactate; ▵, acetate; ▴, Fru(1,6)P₂; ▪, G6P; (○), 3-PGA; ◊, PEP.

FIG. 2.

Series of ¹³C spectra during glucose metabolism by the NOX⁺ strain. Each spectrum represents 30 s of acquisition. Glucose was added at time zero, and each spectrum was acquired during the indicated interval and was processed with 5-Hz line broadening. Symbols: ▴, [1-¹³C]Fru(1,6)P₂; ▾, [6-¹³C]Fru(1,6)P₂; ▪, G6P; ○, aspartate.

FIG. 3.

(A to D) Kinetics of [6-¹³C]glucose consumption and product formation (A and B) and pools of intracellular metabolites (C and D) in the NOX⁺ strain under an oxygen atmosphere (A and C) or under anaerobic conditions (B and D) as determined by ¹³C-NMR. (E) Biochemical parameters determined by ³¹P-NMR during metabolism of glucose in the NOX⁺ strain under aerobic conditions. The shaded area indicates glucose availability. Symbols: ⧫, glucose; □, lactate; ▵, acetate; •, acetoin; ▴, Fru(1,6)P₂; ▪, G6P; ○, 3-PGA; ◊, PEP; ∗, NTP; ▿, P_i; ▾, intracellular pH.

FIG. 3.

(A to D) Kinetics of [6-¹³C]glucose consumption and product formation (A and B) and pools of intracellular metabolites (C and D) in the NOX⁺ strain under an oxygen atmosphere (A and C) or under anaerobic conditions (B and D) as determined by ¹³C-NMR. (E) Biochemical parameters determined by ³¹P-NMR during metabolism of glucose in the NOX⁺ strain under aerobic conditions. The shaded area indicates glucose availability. Symbols: ⧫, glucose; □, lactate; ▵, acetate; •, acetoin; ▴, Fru(1,6)P₂; ▪, G6P; ○, 3-PGA; ◊, PEP; ∗, NTP; ▿, P_i; ▾, intracellular pH.

FIG. 4.

(A and B) Kinetics of [1-¹³C]glucose consumption and product formation and pools of intracellular metabolites in the NOX⁻ strain under an oxygen atmosphere (A) and under anaerobic conditions (B) as determined by ¹³C-NMR. (C) Evolution of the pyridine nucleotides before and after addition of [1-¹³C]glucose (80 mM) under anaerobic conditions. For these experiments, pyridine nucleotides were enriched in ¹³C by cultivation of the NOX⁻ strain in medium containing ¹³C-labeled nicotinic acid. Symbols: ⧫, glucose; □, lactate; ▵, acetate; ▴, Fru(1,6)P₂ (FBP); ○, 3-PGA; yellow inverted triangles, NAD⁺; blue circles, NADH.

FIG. 4.

(A and B) Kinetics of [1-¹³C]glucose consumption and product formation and pools of intracellular metabolites in the NOX⁻ strain under an oxygen atmosphere (A) and under anaerobic conditions (B) as determined by ¹³C-NMR. (C) Evolution of the pyridine nucleotides before and after addition of [1-¹³C]glucose (80 mM) under anaerobic conditions. For these experiments, pyridine nucleotides were enriched in ¹³C by cultivation of the NOX⁻ strain in medium containing ¹³C-labeled nicotinic acid. Symbols: ⧫, glucose; □, lactate; ▵, acetate; ▴, Fru(1,6)P₂ (FBP); ○, 3-PGA; yellow inverted triangles, NAD⁺; blue circles, NADH.

FIG. 5.

Schematic representation of glucose metabolism under aerobic and anaerobic conditions in L. lactis. Major regulation sites described in the text are highlighted. The different sizes of the letters used for intracellular compounds reflect the different concentrations. FBP, fructose bisphosphate; GAP, glyceraldehyde 3-phosphate.