(41d) Engineering Auto-Regulatory Time-Dependent Genetic Circuits for Improved Biochemical Production in Escherichia Coli

Escherichia coli, a common industrial workhorse, can be engineered to perform biocatalysis of lignocellulosic feedstock into valuable products. However, issues such as cell viability, substrate toxicity and product release often plague the efficiency of bioconversion. In this study, to overcome these challenges, we aimed to develop a range of novel genetic circuits that enable auto-regulatory time-dependent production of biochemicals in E. coli, To improve cell viability, we implemented the control theory in protein expression through the introduction of bio-sensor with time-delay element. The time-delay element ensures that the host cells would not be induced at early stage of growth. This time-delay biosensor not only eliminates the need for costly inducers such as arabinose and IPTG, but it also allows optimal performance in dynamic biological system through corrective actions. As proof of concept, we designed and installed a time-delay biosensor to enable E. coli to detect hydroxycinnamic acid and convert it to valuable biochemicals. Further, to counter the problem of substrate toxicity, we performed strain optimization using hydroxycinnamic acid as our test substrate, in which the improved strain was capable of tolerating up to 2.5-fold of the previously accepted concentration. Lastly, to aid in the extraction of biochemical products and simplify the product harvest process from E. coli, we developed a novel genetic circuit that conferred cell density-dependent auto-regulatory lysis. We envision that our auto-regulatory time-dependent approach can be readily extended to improving production of a range of biochemicals in E. coli.