Metabolic flux distribution in a metabolically engineered Escherichia coli strain producing succinic acid

Escherichia coli NZN111, which is known as a pfl ldhA double mutant strain, was metabolically engineered to produce succinic acid by overexpressing melic enzyme into the E. coli controlled by a trc promoter. Fermentation studies were carried out in a LB medium by first growing cells aerobically to an OD600 of 5. At this point, 0.01 mM IPTG was added to induce the overexpression of malic enzyme and the agitation speed was gradually lowered. When the culture OD600 reached 11, a complete anaerobic condition was achieved by flushing with a CO2-H-2 gas mixture. When NZN111 (pTrcML) was cultured at 37 degrees C, the final succinic acid concentration of 2.8 g/l could be obtained after 30 h of anaerobic cultivation. The fermentation results were analyzed by the calculation of metabolic fluxes. Metabolic flux analysis showed that about 85% of phosphoenolpyruvate (PEP) was converted to pyruvate. and further converted to malic acid by melic enzyme.