(174bg) Engineering the Transcriptional Repressor-Based Genetic Inverter for the Regulation of Tryptophan Derivatives Production in Escherichia coli

To optimize cellular metabolism for efficient microbial production, genetic circuits with diverse functionalities are developed for achieving intricate regulation. Transcriptional repressors are widespread transcriptional factors, but rarely developed and applied to metabolic regulation due to their single repression functionality. To expand their functionality and improve their applicability, we proposed a strategy to construct a transcriptional repressor-based genetic inverter by integrating two transcriptional repressors. As a proof of concept, we adopted the tryptophan-inducible repressor PtrpO1-TrpR1 and PtetO1-TetR repression system to build a tryptophan-inducible genetic inverter. Within this genetic inverter, PtrpO1-TrpR1 governed the tryptophan detection while PtetO1-TetR was responsible for the expanded activation output and was under the control of PtrpO1-TrpR1. In the presence of tryptophan, the TrpR1-tryptophan complex bound to the PtrpO1 promoter and repressed TetR expression. As TetR abundance decreased, the repression effect exerted by TetR on PtetO1 was attenuated, thereby leading to the activation output. After characterizing and optimizing the inverter, we further coupled the original repression function of PtrpO1-TrpR1 to derive a bifunctional genetic circuit to realize the function expansion of the transcriptional repressor. To explore the potential of the genetic inverter in microbial production, a tryptophan-triggered activation system and a tryptophan-triggered bifunctional system were successfully built for real-time tryptophan production monitoring. Furthermore, both systems were applied to the regulation of tryptophan derivatives production. The tryptophan-triggered activation system was employed for the dynamic regulation of tryptamine production. Additionally, the tryptophan-triggered bifunctional regulation system was employed for the dynamic regulation of violacein production. Aided by these regulation systems, we successfully enhanced the titers of tryptamine and violacein by 2.0 and 7.4 folds, respectively. This study expanded the functionality of the transcriptional repressor and demonstrated the outstanding performance of applying the transcriptional repressor-based genetic inverter in biosynthetic pathway regulation.