Using a Biosensor Controlled Genetic Feedback Loop to Improve Microbial Biofuel Tolerance

Advanced biofuels offer a promising alternative fuel source to gasoline and diesel because they are renewable sources of energy that are compatible with existing fuel infrastructures.  Engineered microbes can synthesize these biofuels but can only tolerate limited concentrations in their environment. To increase biofuel yield, and to make biofuels an economically competitive fuel source, microbial biofuel tolerance must be increased. Previous research has shown that efflux pumps are an effective tolerance mechanism for cell growth in biofuels. These pumps are complexes of proteins that identify harmful compounds and remove them from the cell. However, overexpression of these pumps can also inhibit cell growth and, therefore, decrease biofuel production. Biofuel and pump expression toxicity must be balanced with each other in order to maximize cell growth.

Our research goal is to design a synthetic feedback loop that will be able to detect biofuel with a sensor protein, which will then trigger the expression of the efflux pump in the host E. coli. The protein MexR, native to Pseudomonas aeruginosa, was chosen as the biosensor because it detects oxidative stress, such as that caused by certain biofuels, in cells. In the feedback loop, MexR represses the expression of the efflux pump by binding to synthetic promoter regions. To best control the system we have chosen to use an alternative sigma factor, rather than relying on E. coli’s native ones which can respond to environmental stress, such as oxidative stress.  A sigma factor native to Pseudomonas putida was chosen because studies show that it did not interact with two of E. coli’s main sigma factor families.  In order to choose the best synthetic promoter region, we have designed a competition experiment for over 500 promoter regions, to allow our system to thrive in a dynamic environment.