Metabolic engineering of Escherichia coli and in silico comparing of carboxylation pathways for high succinate productivity under aerobic conditions

Abstract

A novel aerobic succinate production system was strategically designed that allows Escherichia coli to produce and accumulate succinate with high specific productivity under aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhA, iclR) and byproduct formation pathways (poxB, ackA-pta, mgsA) of E. coli were created to construct the glyoxylate cycle and oxidative branch of the TCA cycle for aerobic succinate production. Strain ZJG13 (ΔsdhA, ΔackA-pta, ΔpoxB, ΔmgsA, ΔiclR) exhibited normal growth behavior and accumulated succinate with an average specific productivity of 0.50mmolg CDW(-1)h(-1) during the fermentation. The glyoxylate shunt operon aceKBA was overexpressed by introducing plasmid pT9aceKAB to ZJG13; the resulting strain had minor effect on productivity improvement. To fully understand the effect of the carboxylation reactions on succinate production, three reactions catalyzed by pyruvate carboxylase (PYC), malic enzyme (MAEA) and phosphoenolpyruvate carboxylase (PPC) were analyzed by a Computational Approach for Strain Optimization aiming at high Productivity (CASOP). Based on the CASOP analysis, carboxylation reaction catalyzed by PYC was the most suitable one to obtain high productivity. When pyc was overexpressed in ZJG13, the specific succinate productivity further increased to 0.76mmolg CDW(-1)h(-1). Fed-batch culture of the strain ZJG13/pT184pyc led to a titer of 36.1g/L succinate, with a specific productivity of 2.75mmolg CDW(-1)h(-1) which stands for the highest value among currently reported aerobic bacterial succinate producers. These results indicate that the CASOP strategy is useful as a guiding tool for the rational strain design with high productivity.

Keywords: Aerobic conditions; Escherichia coli; In silico; Metabolic engineering; Succinate.