LAUSR

Microorganisms can produce hydrocarbons that can serve as replacements or additions to conventional liquid fuels for use in the transportation sector. However, a common problem in the microbial synthesis of biofuels is that these compounds often have toxic effects on the cell. In this study, we focused on mitigating the toxicity of the biojet fuel precursor pinene on Escherichia coli. We used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP.putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP.putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP.putida, while others provide tolerance across a wide range of rpoDP.putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries.