Architecting a Transcriptional Repressor-based Genetic Inverter for Tryptophan Derived Pathway Regulation in Escherichia coli

Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, PtrpO1-TrpR1 and PtetO1-TetR. In this genetic inverter, the repression of TetR towards PtetO1 could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of PtrpO1-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.