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

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