Abstract: To rapidly develop tryptophan overproducers, high-throughput screening was performed on key enzyme in the tryptophan biosynthetic pathway and the chassis strains. The tube fermentation system was optimized to maintain high production consistency with flask fermentation. A colorimetric method compatible with the tube fermentation system was established for tryptophan quantification, using the reaction of tryptophan with naphthylethylenediamine dihydrochloride, providing a detection range of 50 mg/L to 1000 mg/L. Using this method, a superior enzyme mutant, TktA2, was successfully identified from a random mutagenesis library of transketolase TktA. Compared to the wild-type TktA, TktA2 exhibited a more compact dimer structure and a larger substrate-binding pocket, which contributed to the enhanced enzyme stability and substrate turnover rate. Additionally, a dominant chassis strain, T51, was isolated from a genome-reduced strain library of MG1655. Overexpression of TktA2 in T51 yielded the tryptophan overproducer, T51A2ΔtrpRΔtnaB, producing 31.37 g/L tryptophan in a 3 L bioreactor, at a sugar-to-acid conversion rate of 19.01% and an 80.49% higher titer than that of the parental strain. This employed a rapid tryptophan detection method for cost-effective and efficient screening of key enzyme and chassis strains at the tube fermentation level. The integrated high-yielding tryptophan producer maintained high-performance even after fermentation scale-up, laying a foundation for the development of tryptophan overproduction strains.

Key words: tryptophan overproducers, tube fermentation, chassis screening, enzyme screening, tryptophan-colorimetric method