Lawrence Berkeley National Laboratory

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (micro-aerobic) chemostat conditions using 13C labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both GC-MS and 13C-NMR. Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were mainly utilized for biomass synthesis (flux below 5 percent of the lactate uptake rate). The anapleurotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount of carbon was oxidized via the highly reversible serine metabolic pathway. Fluxes through the TCA cycle were less whereas acetate production was more under oxygen limitation than under carbon limitation. Although flux distributions under aerobic, micro-aerobic, and shake-flask culture conditions were dramatically different, the relative flux ratios of the central metabolic reactions did not vary significantly. Hence, S. oneidensis metabolism appears to be quite robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C labeled substrates and to obtain more accurate flux values.