Elucidating the central carbon metabolism of Bacillus megaterium QM B1551
We investigated the central carbon metabolism of Bacillus megaterium QM B1551 cells using various carbon substrates, long-term isotopic enrichment experiments with 13C-labeled carbons, metabolic flux analysis (MFA), and kinetic incorporation of 13C-labeled carbons. We determined that gluconate can be incorporated into the metabolic network structure of B. megaterium QM B1551, particularly when gluconate was present in the environment. It was shown in the same experiment that B. megaterium QM B1551 did not use the Entner-Doudoroff (ED) pathway for glucose (Gluc) metabolism, and instead relied primarily on the Embden-Mayerhof-Parnas (EMP) and oxidative pentose phosphate (PP) pathways. The TCA cycle of this bacterium was incompletely bifurcated, although this bifurcation did not impede the ability of the bacterium to grow. There were bottlenecks in the fluxes from metabolite nodes alpha-ketoglutarate (αKG) to succinate, from succinate to fumarate, and from oxaloacetate (OAA) to fumarate. This bifurcation was overcome with a direct input of substrate into the TCA cycle. The stereochemistry of citrate formation and cleavage was different compared to other bacterial flux studies. It was hypothesized that the carboxylation reaction which combines CO2 and pyruvate (PYR) added CO2 behind the third carbon position of PYR to form OAA. It was further hypothesized that OAA was flipped such that the fourth carbon position of OAA became the fourth carbon position of citrate. Finally, it was observed that B. megaterium QM B1551 had an early preference for glutamate catabolism in the TCA cycle, followed by a preference for Gluc catabolism across the metabolic network. These findings provide clarification regarding contradictions in the literature, and thus contribute towards finding new platforms for optimized industrial use of this bacterium.
Metabolic flux analysis; Bacillus megaterium; 13C isotopic labeling
dissertation or thesis