Figure 1.
Monitoring of M. extorquens AM1 growth parameters in batch-culture with 60 mM methanol plus 15 mM succinate.
A. Monitoring of Optical Density (OD) at 600 nm (black circle), methanol consumption (black triangle) and succinate (white triangle) consumption. B. Monitoring of oxygen partial pressure (pO2) (white circle), 13C (black square) and 12C (white square) CO2 production in exhaust gas. Metabolite sampling was performed at three timepoints as indicated in A: mid-co-consumption phase (Sampling time 1, S1), end co-consumption phase (Sampling time 2, S2), and transition phase (Sampling time 3, S3). The figure shows data of one replicate.
Table 1.
Growth parameters of M. extorquens AM1 cells growing in batch-culture in minimal medium with 120 mM methanol, or 15 mM succinate, or 60 mM methanol plus 15 mM succinate.
Figure 2.
Average 13C labeling in intra-cellular metabolites measured by LC-MS during the growth of M. extorquens AM1 upon co-consumption with 13C (>99%) methanol and natural abundance (1.1% 13C) succinate.
Metabolite quenching, extraction and measurements were performed specifically for each class of metabolite, i.e., amino acids, polar compounds, and coenzyme A thioesters, as described in the materials and methods. Average 13C labeling. (black): Sample collected during mid-co-consumption phase (Sampling time 1 in Figure 1), (gray hatched): sample collected at the end of the co-consumption phase (Sampling time 2 in Figure 1).
Figure 3.
Central metabolic network map displaying selected mass isotopomer distributions of the central metabolites measured by LC-MS of M. extorquens AM1 growth upon co-consumption with 13C (>99%) methanol and natural abundance (1.1% 13C) succinate.
The precursors of the amino acids measured in the central metabolism and directly measured metabolites are indicated in boxes. The box colors indicate substrate-specific carbon incorporation: orange from succinate, blue from methanol, green from both. Mass isotopomer data correspond to samples collected during the mid-co-consumption phase (Sampling time 1 in Figure 1).
Figure 4.
Prediction of the mass isotopomer distribution of hexose-phosphate depending of the M1 mass isotopomers fraction into C3 precursors of gluconeogenesis.
Black, predicted values; red, measured values. The mass isotopomer fraction of hexose-phosphate measurements were 0.63±0.017 for M0, 0.32±0.019 M1, 0.05±0.020 M2, and 0.002±0.020 M3. These values correspond to the probabilistic recombination of two C3-units from gluconeogenesis assuming that 20% of the C3 units have incorporated one 13C carbon and 80% of the units are not labeled.
Figure 5.
Metabolic contribution of succinate and methanol in energetics (ATP, NADH, NADPH) during co-consumption calculated by metabolic flux analysis through the genome-scale metabolic network of M. extorquens AM1 [20].
Methanol and succinate contributions are indicated in black and white, respectively, for a feasible solution under the constraints of the measured experimental values (methanol uptake rate, succinate uptake rate, growth rate, CO2 production rate). The % of their contribution to all needs is indicated at the left part of the bars; the values under the brackets are the lower and upper limits of their contributions calculated by the Flux Variability Analysis. N-GAM: non-growth-associated maintenance; GAM: growth-associated maintenance; Bios.: biomass biosynthesis; Prec. Bios.: precursor biosynthesis through the central metabolic network.