Engineering solvent tolerant microbes

Many petrochemical compounds that are being considered for microbial production are solvent-like in nature. Examples of such compounds are fuels and other bulk chemicals such as precursors for polymers and plastics. For these compounds, two aspects impede the efficiency of microbial production. One is their inherent solvent like nature that results in toxicity towards the microbe. Second is product inhibition due to intracellular accumulation. Therefore the efficiency with which the final product can be exported from the cell has a significant influence on production titer. We have investigated the role of cellular transporters in improving biofuel tolerance and production in Escherichia coli. Using simple but effective competition based strategies we identified pumps that bestowed tolerance against representative biogasoline, biodiesel and biojetfuel candidates. In cases where the targeted approach was not possible, we used functional genomics data to prospect for tolerance bestowing transporters. For the compounds where effective transporters were found, expression of the pumps also resulted in improved production levels. Building upon the discovery of these transport systems, we have explored the limits and challenges of using membrane-based mechanisms in host engineering. We are exploring several avenues to optimize the use of transporters for host engineering. Strategies that improve the efficiency of a transporter allow us to bypass the necessity to overexpress these high burden systems. Engineering the host microbe membrane may allow increased use of the pertinent transporters. Optimization of expression systems has also proven important in maximizing the benefits from a specific pump in a fuel production strain.