Mitigating Host Burden of Genetic Circuits by Engineering Autonegatively Regulated Parts and Improving Functional Prediction

ABSTRACT: Mitigating unintended interferences between circuits and host cells is key to realize applications of synthetic regulatory systems both for bacteria and mammalian cells. Here, we demonstrated that growth burden and circuit dysregulation occurred in a concentration-dependent manner for specific transcription factors (CymR*/CymR) in E.coli, and direct negative feedback modules were able to control the concentration of CymR*/CymR, mitigate growth burden, and restore circuit functions. A quantitative design scheme was developed for circuits embedded with autorepression modules. Four key parameters were theoretically identified to determine the performance of autoregulated switches and were experimentally modified by fine-tuning promoter architectures and cooperativity. Using this strategy, we synthesized a number of switches and demonstrated its improvement of product titers and host growth controlling the complex deoxyviolacein biosynthesis pathway. Furthermore, we restored functions of a dysregulated multilayer NOR gate by integrating autorepression modules. Our work provides a blueprint for engineering host-adaptable synthetic systems.

Automated summary

This study focuses on mitigating unintended interferences between circuits and host cells and proposes a design scheme to control concentration through feedback modules. By adjusting promoter architectures and cooperativity, several switches were successfully synthesized, improving the product titers and host growth. Furthermore, the functions of a dysregulated multilayer NOR gate were restored through the integration of autorepression modules.