(228bb) Improving Dynamic Regulation of Biodiesel Production in E. coli

Dynamic regulation is frequently employed in nature to optimally allocate cellular resources for efficient substrate utilization. Dynamic regulation is utilized as an advanced metabolic engineering strategy to improve production of a bioproduct by employing metabolite responsive feedback of gene expression. The dynamic nature of these feedback control schemes require a sensor, which detects metabolite concentration in real-time, and an actuator, which regulates gene expression based on the sensor measurements. Bacterial transcription factors have been used for this purpose, as they are sensor-regulators by design. This approach has been implemented for the production of fatty acid ethyl ester (FAEE) biodiesel in E. coli with great success. Based on a tractable non-steady model of this pathway, we propose to improve the dynamically regulated FAEE production by: 1) altering the ethanol-producing enzymes for more balanced pathway flux, and improving the dynamics of the regulator; 2) increasing the expression of the regulator protein; and 3) engineering a reversible ethanol pathway to enable reutilization of excess ethanol. These changes result in better carbon distribution and bring the pathway into better balance, allowing the dynamic nature of dynamic control schemes to be utilized. The strategies employed here are likely to be generally applicable to metabolic pathways rather than merely specific to this one.