Optimizing L-Tryptophan Production in Escherichia coli through Redox Balancing and Metabolomics Analysis.

L-tryptophan (L-trp) is a key aromatic amino acid with significant industrial value, and microbial fermentation provides a sustainable alternative to traditional chemical synthesis. However, low production yields due to inefficient microbial strains remain a major challenge. In this study, we enhanced L-trp production through redox engineering of Escherichia coli TX1. Metabolomics analysis at various fermentation stages revealed dynamic changes in the metabolites of the aromatic amino acid pathway. A key bottleneck was identified in the shikimate pathway, where significant accumulation of chorismate and shikimate led to inefficient L-trp production. By optimizing the shikimate pathway, L-trp production was increased by 19.8%. Additionally, the continuous accumulation of phosphoenolpyruvate suggested a limitation in the supply of erythrose-4-phosphate, which participates in the same reaction. Redirecting carbon flux from fructose-6-phosphate toward erythrose-4-phosphate increased the precursor pool of erythrose-4-phosphate. To overcome nutritional limitations, exogenous addition of amino acids, vitamins, and salt ions to the fermentation medium was implemented. Systematic metabolic engineering and fermentation optimization led to a significant improvement in tryptophan production, achieving an 86.6% increase compared to the original level. This study lays a solid foundation for the future development of more efficient tryptophan-producing strains.