Biosensor-driven evolution and metabolic engineering of an Escherichia coli cell factory for L-tryptophan production.

L-Tryptophan is an important aromatic amino acid with wide applications across the food, pharmaceutical, and feed industries. However, its efficient microbial production remains challenging due to complex metabolic networks and multi-level feedback regulation. In this study, we constructed a highly efficient Escherichia coli cell factory for L-tryptophan biosynthesis by combining systematic metabolic engineering with high-throughput screening. Initially, a tnaC-based biosensor was developed and coupled with atmospheric and room temperature plasma (ARTP) mutagenesis to isolate high-performance chassis strains. Central carbon metabolism was subsequently reprogrammed to minimize carbon loss and channel metabolic fluxes toward essential precursors, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P). To further alleviate pathway bottlenecks, promoter engineering was utilized to balance the intracellular supplies of L-glutamine, L-serine, and phosphoribosyl pyrophosphate (PRPP). This targeted intervention yielded a 21.61% increase in L-tryptophan accumulation. Product transport systems were then engineered to enhance extracellular secretion and mitigate intracellular toxicity. Following the optimization of inoculum size and feeding strategies in a 5 L bioreactor, the final engineered strain (W-24) produced 50.83 g/L of L-tryptophan within 40 h, achieving a yield of 0.185 g/g glucose. This multi-modular engineering framework establishes a robust platform for L-tryptophan biosynthesis and provides a scalable strategy for the industrial production of other valuable aromatic compounds.