Engineering Auto-Regulatory Genetic Circuits That Enable Modular and Programmable Functionalities for Biotechnological Applications

Engineering auto-regulatory genetic circuits that enable modular and programmable functionalities for biotechnological applications

Tat-Ming Lo1,2 and Matthew Wook Chang1,2

1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore 117597

2Synthetic Biology Program for Clinical and Technological Innovation, National

University of Singapore, Singapore 117456
Microbes can be reprogrammed to demonstrate biotechnologically important functionalities such as biochemicals production. However, reprogramming microbes to perform complex and coordinated tasks often requires the use of additional exogenous chemicals such inducers, which can increase production costs, introduce host toxicity and incompatibilities with industrial scale-up. To overcome this challenge, we developed auto-regulatory genetic circuits by introducing novel sensors and feedback loops into microbial systems. Not only our engineered systems are independent of costly inducers, they are capable of regulating protein expression based on desired features such as substrate concentration and cell-density. One example is the use of our genetic circuit in auto-regulatory lignocellulosic bioconversion. In this system, the genetic circuit was engineered to regulate enzyme expression based on lignocellulose substrate cues and cell-density. In another example, we used our cell density-dependent circuit to facilitate product extraction in Escherichia coli. Macromolecular product extraction in E. coli often requires chemical, enzymatic, and/or mechanical lysis, thus increasing production costs. To aid in the extraction of biochemical products and simplify the product harvest process, we developed a genetic circuit that enabled cell density-dependent auto-regulatory lysis based on a transducer-switch scheme. As a proof of concept, we demonstrated the applicability of the lysis circuit to biotechnological applications by employing plasmid DNA extraction as a test bed. We envision that our engineered auto-regulatory circuits that enable programmable functionalities can be readily applied to a range of biotechnological applications.