Engineering Tryptophan Synthase for Efficient Bioconversion of l-Tryptophan in Escherichia coli

l-Tryptophan, an essential amino acid, serves as a critical precursor for the biosynthesis of numerous bioactive compounds. In this study, we engineered tryptophan synthase (TrpS) to develop an efficient cell factory for l-tryptophan production. Initially, EcTrpS from Escherichia coli K-12 was expressed in E. coli BL21 (DE3) to construct strain BS, which produced 836.43 mg/L of l-tryptophan. To alleviate substrate inhibition, we constructed a mutant library of 104–105 variants through two rounds of error-prone PCR and utilized a biosensor employing a green fluorescent protein to report l-serine consumption for screening TrpS mutants. Mutant C10 showed a 375% increase in enzyme activity compared with strain BS. Computational analysis was used to explore an improved catalytic mechanism. Finally, through systematic process optimization and catalytic strategy, strain C10 produced 94.2 g/L of l-tryptophan, the highest titer via whole-cell transformation. This study provides new perspectives and strategies for TrpS engineering and the industrial production of l-tryptophan.