The market of L-phenylalanine has been stimulated by the great demand for the low-calorie sweetener aspartame. In this paper, the effects of pivotal genes on L-phenylalanine production were evaluated by metabolic engineering of wild type Escherichia coli. The bifunctional PheA protein contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by L-phenylalanine). The catalytic domain of PheA was overexpressed to increase L-phenylalanine production. It was firstly indicated that this domain could enhance the metabolic influx to overproduce L-phenylalanine and improve the survival ability under m-Fluoro-DL-phenylalanine stress. Furthermore, the fermentation performance of aroG feedback inhibition resistant mutants was firstly compared, aroG29 and aroG15 increased the L-phenylalanine concentration by 5-fold. After that the expression of aroK and ydiB was also elevated, and the L-phenylalanine yield on cell (0.79 g/g) and maximum L-phenylalanine productivity (0.073 g/L/h) were subsequently doubled. Meanwhile, the L-phenylalanine yield on glucose increased from 0.124 g/g to 0.153 g/g. It was found that genes ydiB and aroK could elevate the L-phenylalanine yield and productivity and shorten the lag phase. (C) 2013 Elsevier Ltd. All rights reserved.