L-Phenylalanine, a critical intermediate in the food and pharmaceutical industries, requires enhanced biosynthetic efficiency to meet industrial demands.This study engineered the L-phenylalanine biosynthetic pathway in Escherichia coli W3110 through systematic metabolic modifications.Initially, the regulatory genes lacI and tyrR were deleted to alleviate metabolic repression, while the feedback inhibition-resistant variant aroGfbr was overexpressed to reinforce the supply of the precursor DAHP.Subsequently, inactivation of csrA and poxB optimized carbon flux distribution, achieving simultaneous improvements in biomass accumulation and substrate utilization efficiency.Further metabolic optimization involved chromosomal integration of the feedback-resistant pheAfbr, ilvE, and aromatic biosynthesis module genes (aroB, aroD, aroK, aroC), coupled with deletion of the competitive pathway gene ydiB, to reconstruct a streamlined central metabolic network.Finally, integration of the transporter gene yddG and deletion of aroP enhanced product secretion, yielding the engineered strain M13.Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed the enhanced mRNA abundance of key genes post-engineering.In a 5 L fed-batch fermentation system, strain M13 produced 62.7 g/L L-phenylalanine with a glucose-to-product conversion yield of 22.3% after 60 h.This study elucidated the mechanistic impact of sequential metabolic engineering on L-phenylalanine biosynthesis, with transcriptional profiling revealing the regulatory effects of genetic modifications on metabolic flux distribution.These findings provide critical data for overcoming bottlenecks in L-phenylalanine biosynthesis and establish a methodological framework for developing high-yield microbial platforms for aromatic amino acids and their derivatives.
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