Error-prone PCR of the tryptophan synthase coding gene in Escherichia coli to enhance the affinity for indole under inhibitory concentrations

Abstract

Tryptophan synthase (TrpS), a key enzyme for l-tryptophan biosynthesis, catalyzes the conversion of indole and L‑serine into l-tryptophan via the synergistic action of its α subunits (encoded by trpA) and β subunits (encoded by trpB). However, the suboptimal catalytic efficiency of the wild-type enzyme poses a major bottleneck for industrial l-tryptophan production. In this study, we employed error-prone PCR to mutate the TrpS-coding gene and a mutant with enhanced affinity for indole was screened using an l-tryptophan fluorescence sensor. Sequencing and alignment revealed 23 mutated residues in this mutant. To identify the key amino acids that affect the affinity of TrpS for indole, these 23 mutated residues were individually mutated in TrpS-coding gene from l-tryptophan-producing strain JW-5 by site-directed mutagenesis. The experimental results demonstrate that the mutants TrpS(B)^K61A, TrpS(B)^G84S, TrpS(B)^Q114M, and TrpS(B)^F385V significantly enhance the production of l-tryptophan. Subsequently, these effective residues were combined, and the results indicated that the TrpS(B)^Q114M,G84S showed as the optimal mutant. TrpS(B)^Q114M,G84S exhibited the improved k_cat (s^-1) and the reduced k_m (μM) for indole compared to the wild-type, indicating significantly enhanced the affinity for indole. Molecular docking analyses revealed that the substitutions at positions 114 (glutamine to methionine) and 84 (glycine to serine) restructured the binding pocket of TrpS, resulting in stronger interactions with indole. Scale-up fermentation in a 5 L bioreactor confirmed the efficacy of the engineered strain. After 40 h of fed-batch fermentation, the mutant strain achieved an l-tryptophan titer of 34.43 g/L, a 61.03 % increase over the control strain JW-5 (21.38 g/L). In addition, the conversion yield improved from 14.8 % to 21.6 %. These findings demonstrated that the substitutions at positions 114 and 84 in the β-subunit of TrpS significantly enhance indole affinity, thereby boosting l-tryptophan production and conversion efficiency.