Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures

Abstract

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.

FIG. 1.

Purine and riboflavin biosynthesis pathway. Produced or consumed cofactors or building blocks are shown below the pathway. Abbreviations: P5P, pentose-5-phosphate; PRPP, phosphoribosylpyrophosphate; ∼P, number of equivalents.

FIG. 2.

The metabolic flux state is one determinant of the isotopomer pools in metabolic intermediates and amino acids. Additionally, the isotopomer pools are influenced by the factors shown in gray. By use of isotopomer balancing, the metabolic flux state may be estimated (broken arrows) from NMR- or mass spectroscopy-based isotopomer data and knowledge of the additional influential factors (shown in gray).

FIG. 3.

Specific uptake rates for glucose, cofed substrates, and total carbon in carbon-limited chemostat cultures. Cultures were grown either on glucose only or on mixtures of glucose and the indicated cosubstrates.

FIG. 4.

Biomass yields and riboflavin yields in chemostat cultures. The biomass yields are calculated per mole of carbon (left, dark gray bars), mole of electrons (middle, light gray bars), or mole of ATP (right, black bars) available in the substrates.

FIG. 5.

Metabolic flux distribution of B. subtilis RB50::[pRF69]_n in carbon-limited chemostat cultures with glucose (A), glucose and gluconate (B), glucose and acetate (C), glucose and acetoin (D), and glucose and citrate (E). D was 0.1 h⁻¹ (C and E) or 0.12 h⁻¹ (A, B, and D). Gray arrows indicate precursor withdrawal for biomass biosynthesis. Net and exchange fluxes (millimoles gram⁻¹ hour⁻¹) are given in square and oval boxes, respectively. Due to the fitting procedure used for intracellular flux estimation, extracellular fluxes can show small deviations from the experimentally determined data (Fig. 3). Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PG, 6-phophogluconate; E4P, erythrose-4-phosphate; T3P, triose-3-phosphate; PGA, phosphoglycerate; ACA, acetyl-CoA; OGA, oxoglutarate; MAL, malate; FUM, fumarate; CIT, citrate; n.d., not determined.

FIG. 5.

Metabolic flux distribution of B. subtilis RB50::[pRF69]_n in carbon-limited chemostat cultures with glucose (A), glucose and gluconate (B), glucose and acetate (C), glucose and acetoin (D), and glucose and citrate (E). D was 0.1 h⁻¹ (C and E) or 0.12 h⁻¹ (A, B, and D). Gray arrows indicate precursor withdrawal for biomass biosynthesis. Net and exchange fluxes (millimoles gram⁻¹ hour⁻¹) are given in square and oval boxes, respectively. Due to the fitting procedure used for intracellular flux estimation, extracellular fluxes can show small deviations from the experimentally determined data (Fig. 3). Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PG, 6-phophogluconate; E4P, erythrose-4-phosphate; T3P, triose-3-phosphate; PGA, phosphoglycerate; ACA, acetyl-CoA; OGA, oxoglutarate; MAL, malate; FUM, fumarate; CIT, citrate; n.d., not determined.

FIG. 6.

Specific rates of NADPH production and consumption in carbon-limited chemostat cultures. NADPH production is accomplished via the oxidative PP pathway (lower, dark gray bars) and isocitrate dehydrogenase (upper, light gray bars). NADPH consumption occurs via biomass formation, transhydrogenase, and riboflavin biosynthesis (dark gray, light gray, and black bars, respectively).

FIG. 7.

Specific ATP production rates in carbon-limited chemostat cultures. The ATP fraction consumed for biomass (including riboflavin) formation and futile cycling via malic enzyme and PEP carboxykinase and the excess ATP fraction are indicated by light gray, black, and dark gray bars (top to bottom), respectively.

FIG. 8.

Cellular contents of ATP (left, light gray bars) and ADP (right, dark gray bars) in carbon-limited chemostat cultures. CDW, cell dry weight.