Rational metabolic engineering has numerous applications in the optimization of microorganisms for the production of valuable compounds at the laboratory-scale. However, the existing strategies and tools are far from sufficient for engineering of industrial strains due to their specificity. The aim of this project was to implement novel strategies to enhance industrial l-phenylalanine (l-PHE) production and yield, including the regulation of key gene expressions, modifications of global transcription factors, creation of NADPH-independent pentose phosphate pathway and pyruvate-oxaloacetate-phosphoenolpyruvate cycle. The project also involved the identification and engineering of novel byproduct pathways and the development of a tyrosine-nonauxotrophic strain. Through comprehensive rational engineering, an industrial l-PHE producer, designated PHE17, achieved the highest production (103.15 g/L) and yield (0.229 g/g) of l-PHE reported thus far. This study also represents the first report on the iterative engineering of industrial l-PHE producers, thereby offering great significance for the engineering of other aromatic animo acids-producing strains.