Development of Metabolite-Responsive Transcription Factor Systems as Modular Platforms for Gene Expression Control

Traditional inducible systems typically induce the simultaneous expression of all genes controlled by similar promoters, thereby limiting their use. In this study, we used two metabolite-inducible systems, MarR from the <i>Escherichia coli mar</i> operon and TtgR from the <i>Pseudomonas putida ttg</i> operon, to assess their use as gene regulation platforms beyond reporter assays. Ligand-dependent transcription was validated using eGFP. The reporter was replaced with two flavonoid O-methyltransferases (OMTs), ROMT-9 and SOMT-2, under transcription factor (TF)-specific promoters. In <i>E. coli</i>, both systems enabled in using HPLC. TF-based expression did not impact enzyme activity. Induction with salicylic acid (MarR) produced stronger gains than that with 4'-hydroxyflavanone (TtgR), although the overall fold-changes in product levels were regulated by basal (leaky) expression. Thus, although transcriptional control was robust, enzymatic regulation was less stringent, highlighting the necessity for genetic engineering of components, including TFs, promoters, transcription factor binding sites, and ribosome binding sites, to reduce leakiness and expand the dynamic range. Overall, these orthogonal and modular TF-based systems offer a framework for independent and inducible control of multiple genes, with potential applications in biosensing, metabolic engineering, and programmable pathway design.