L-phenylalanine(L-Phe) is an essential amino acid for both humans and animals, with widespread applications in the food, pharmaceutical, and feed industries.With the increasing market demand, problems such as low production efficiency of traditional microbial fermentation methods have been gradually highlighted, so it is necessary to obtain an efficient and stable L-Phe producing strain.In this study, we first constructed an L-Phe-producing Escherichia coli engineering bacterium based on traditional metabolic engineering strategies, and obtained an L-Phe-producing strain by means of lifting the feedback inhibition, optimizing the expression level or activity of key enzymes of the shikimate pathway, optimizing the supply of precursors, such as phosphoenolpyruvic acid and erythrose 4-phosphate by means of promoter engineering, deactivating the synthesis pathway of the byproduct acetic acid and optimizing amino acid transporter pathway.To further improve production performance, a global transcription machinery engineering(gTME) strategy was employed.This involved constructing a random mutagenesis library for the rpoD gene, which encodes the σ70 factor, and the rpoA gene, which encodes the α subunit of RNA polymerase, as well as developing an L-Phe biosensor-based detection circuit to screen for high-yield mutants.The engineered strain PHE-56 achieved an L-Phe titer of 79.68 g/L, a productivity of 1.66 g/(L·h), and a yield of 22% in a 5 L fermenter after 48 hours of fed-batch fermentation.This study combines multiple metabolic engineering strategies, demonstrating the efficacy of gTME in metabolic engineering, providing a new perspective for the development of an L-Phe cell factory, and offering a promising strain platform for the industrial production of L-Phe.
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