Construction and anaerobic fermentation of metabolically engineered Escherichia coli producing succinate

To study the effect of malic enzyme overexpression on succinate production in the pfl ldh double mutant Escherichia coli NZN111 (ldhA:: Kan pfl:: Cam) , we transformed the expression vector pTrc99a-sfcA into it and constructed the recombinant NZN111(pTrc99a-sfcA). The specific malic enzyme activity of the recombinant was 30.67 u/mg after 8-hour inducement by 0.5 mmol/L Isopropyl β-D-1-Thiogalactopyranoside, 140 times higher than that of NZN111. The two-step fermentation was used and the results showed that the overexpression of malic enzyme catalyzed the reverse reaction from pyruvate to malate, which was impossible under general conditions. Succinate accumulated as the major product. Cells at the late exponential phase were inoculated to an anaerobic fermentation with 0.7 mmol/L IPTG in medium containing 18.5 g/L glucose. The final concentration of succinate and acetate was 12.84 g/L and 0.58 g/L, respectively. Formate and lactate were not detected. The constructed metabolically engineered strain had the feature of higher succinate yield and less by-products.