The Tryptophan-Induced Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase () operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and host factors on induction of the leader sequence. To do so, we constructed and biochemically validated an experimental assay using the operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (, l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the gene from strains intended for tryptophan-sensing utilizing the leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.