A systems level engineered E. coli capable of efficiently producing L-phenylalanine

• Inactivation of crr and tyrA increased the yield of L-phenylalanine (Phe).
• Uninhibited manner aroG15 showed thermostability and the highest Phe concentration among all the aroG mutants.
• The genetic switch of gene expression was used to precisely control biomass accumulation and Phe synthesis.
• Jointly expressing yddG and tyrB shifted equilibrium towards Phe synthesis and remited the feedback regulation.
• Engineering of Phe producer was done to illustrate the effectiveness of systematic level engineering.

The biosynthesis of L-phenylalanine (Phe) is one of the most complicated amino acid synthesis pathways. In this study, the engineering of Phe producer was carried out to illustrate the effectiveness of systems level engineering: (1) inactivated glucose specific phosphoenolpyruvate-carbohydrate phosphotransferase (PTS) system by inactivation of crr to moderate the glucose uptake rate to reduce overflow metabolism; (2) genetic switch on or off the expression of phefbr, aroG15, ydiB, aroK, and tyrB to increase the supply of precursors; (3) employed a tyrA mutant strain to reduce carbon diversion and to result in non-growing cells; (4) enhanced the efflux of Phe by overexpressing yddG to shift equilibrium towards Phe synthesis and to release the feedback regulation in Phe synthesis. The mutants in PTS were firstly compared and a crr− mutant was firstly screened. The mutant AroG15 was demonstrated to a thermostable mutant. The strains expressing yddG excreted Phe into the medium at higher rate and less intracellular Phe accumulated. By systems level engineering, an engineered Phe producer achieved 47.0 g/L Phe with a yield of 0.252 g/g which was the highest under the non-optimized fermentation condition.