l-tryptophan, Corynebacterium glutamicum, Metabolic engineering, Transporter engineering, Genome-scale modeling, Biotechnology, TP248.13-248.65, Biology (General), QH301-705.5

Corynebacterium glutamicum is a versatile industrial microorganism for producing various amino acids. However, there have been no reports of well-defined C. glutamicum strains capable of hyperproducing l-tryptophan. This study presents a comprehensive metabolic engineering approach to establish robust C. glutamicum strains for l-tryptophan biosynthesis, including: (1) identification of potential targets by enzyme-constrained genome-scale modeling; (2) enhancement of the l-tryptophan biosynthetic pathway; (3) reconfiguration of central metabolic pathways; (4) identification of metabolic bottlenecks through comparative metabolome analysis; (5) engineering of the transport system, shikimate pathway, and precursor supply; and (6) repression of competing pathways and iterative optimization of key targets. The resulting C. glutamicum strain achieved a remarkable l-tryptophan titer of 50.5 g/L in 48h with a yield of 0.17 g/g glucose in fed-batch fermentation. This study highlights the efficacy of integrating computational modeling with systems metabolic engineering for significantly enhancing the production capabilities of industrial microorganisms.