Engineering of <i>tnaC</i>-Based Tryptophan Biosensors for Dynamic Control of Violacein Production

Tryptophan not only serves as a fundamental building block for protein synthesis but also acts as a metabolic precursor for a diverse array of high-value chemicals. Although a few tryptophan-responsive biosensors are currently available, there is a growing interest in developing high-performance biosensors. In this study, we create a miniature toolkit of tryptophan biosensors based upon the leader regulatory region of the <i>tnaCAB</i> operon, which is responsible for tryptophan catabolism in <i>Escherichia coli</i>. Four variants are generated by engineering the <i>tnaC</i> leader sequence, which encodes a leader peptide composed of 24 amino acid residues. Subsequently, the performance of both the natural <i>tnaC</i> sequence and its engineered variants is assessed in a reporter strain based on the MazEF toxin-antitoxin system. The results demonstrate that two engineered variants exhibit increased sensitivity to low levels of tryptophan. Moreover, the engineered biosensors are further optimized by replacing the native promoter of <i>tnaC</i> with a phage-derived constitutive promoter. Intriguingly, the engineered biosensors can be reconstructed for extended application in <i>Pseudomonas putida</i>, a robust microbial chassis for metabolic engineering. In summary, our study expands the toolkit of tryptophan biosensors that can be broadly used for the bioproduction of many other high-value tryptophan-derived products.