(766g) A Genetic Circuit Approach to Alleviating Membrane Transporter Stress

A key component in microbial production of biofuels and other chemicals is the control of the selective permeability of the cell’s membrane. There is a large research effort in engineering membrane transporters for influx of feed sugars and for efflux of toxic molecules and fermentation products. One limiting factor is the increased stress associated with over-expression of membrane-associated proteins and the difficulty in controlling their expression without the addition of a costly inducer molecule. Therefore expression of transporters must be tightly regulated for maximum productivity and yield. To address this issue, we chose the Escherichia coli protein AcrB, a member of a multi-drug resistance pump, which can be engineered to secrete small biofuel molecules such as alcohols. To control the expression of the somewhat toxic AcrB, we have designed and implemented a feedback circuit. Our circuit utilizes a native E. coli stress promoter that downregulates pump expression in response to stress induced by the pump but not the metabolic product secreted by the pumps. With this presentation, we will discuss how we identified this promoter in a screen of regions upstream of differentially expressed genes in response to various stresses, how the overall pump concentration can be controlled via the ribosome binding site, and how the circuit behaves under various conditions. This approach can also be extended towards stable production of other growth inhibiting or toxic proteins, and can be used to achieve stable, tightly controlled expression, without the drawbacks of small molecule inducer systems.