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

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.