BACKGROUNDAmmonia is a potential neutralizer for bio-based succinic acid production, however, low resistance of Escherichia coli to high concentrations of ammonium inhibit its application. Thus, combined adaptive laboratory evolution (ALE) with pyruvate carboxylase gene over-expression was utilized to obtain an ammonium-tolerant E. coli for efficient succinic acid production. RESULTSMutant E. coliAM060 was obtained by adaptive laboratory evolution. Fermentation results showed that the specific growth rate of recombinant was 0.67-fold faster than BER208. Metabolites analysis of fed-batch fermentation demonstrated that 47gL(-1) glucose was consumed and 27.33gL(-1) succinic acid was produced, which was 0.68- and 0.81-fold higher than with BER208. Key enzyme activities analysis showed that the specific enzyme activities involved in nitrogen assimilation, glucose uptake and ATP supply were all increased. Furthermore, E. coliAM060/pTrc99a-pyc was constructed to reduce the pyruvic acid accumulation and final succinic acid production increased to 33.14gL(-1) with a mass yield of 0.69gg(-1). CONCLUSIONSimultaneous utilization of ALE and a metabolic engineering strategy was effective in obtaining a highly efficient succinic acid-producing strain using ammonia as neutralizer, and the improved ammonium resistance was attributed to the enhanced nitrogen assimilation capability, glucose uptake rate and intracellular ATP supply. (c) 2015 Society of Chemical Industry