Production of succinate by a metabolic engineered Escherichia coli and its scale-up process in fermentor

Abstract:

[Background] Escherichia coli AFP111 produces succinate with low yield from glucose and with high production of acetate. [Objective] To reduce acetate production and improve succinate yield. A 100-L-scale fermentation process was established. [Methods] One-step homologous recombination was used for knocking out the key enzyme gene of acetate synthesis pathway and modifying key enzyme promoter of succinate synthesis pathway. The culture conditions were optimized by using single factor design in a 5-L fermentor. [Results] The resulting strain of SX02, deletion of ackA-pta and tdcDE, reduced acetate production by 53.42%, and increased succinate yield by 9.85% as compared to the original strain. The glucokinase activity of SX03, obtained after over-expressing glk, was 3.66 times high than SX02. Its acetate production reduced by 31.62% and its succinate yield increased by 8.28% as compared to SX02. SX03 produced 3.97 g/L acetate, and 1.62 mol of succinate per mol glucose in a 5-L fermentor with a decrease of acetate production by 75.76% and an increase of succinate yield by 19.12% as compared to the original strain. The fermentation conditions including various neutralizers pH values, agitation speed, glucose concentrations were optimized in a 5-L fermentor. The optimal fermentation conditions were pH 6.8, agitation speed of 250 r/min, glucose concentration of 100 g/L. Under the optimal conditions, SX03 produced 2.24 g/L acetate and 1.66 mol of succinate per mol glucose with a decrease of acetate production by 20.65% and an increase of succinate yield by 2.47%. The fermentation using the strain SX03 was scaled up in a 100-L fermentor, which produced 1.91 g/L acetate and 1.30 mol of succinate per mol glucose. [Conclusion] The metabolic engineered Escherichia coli shows great industrial potential for the commercial production of succinate.